Inhibitors of nucleoside metabolism

ABSTRACT

The present invention provides novel nucleoside-analogue compounds that are effective inhibitors of purine nucleoside phosphorylase (PNP), purine phosphoribosyltransferases (PPRT), and/or nucleoside hydrolases. Also provided are tautomers, esters, prodrugs, and pharmaceutically-acceptable salts of the compounds disclosed herein. The present invention further provides the use of these compounds as pharmaceuticals. The present invention also discloses pharmaceutical compositions containing these compounds. Finally, the present invention provides processes for preparing these compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 09/496,471, filedFeb. 3, 2000, now U.S. Pat. No. 6,228,847, which is a continuation ofU.S. application Ser. No. 09/172,321, filed Oct. 14, 1998, now U.S. Pat.No. 6,066,722, which is a continuation-in-part of U.S. application Ser.No. 08/949,388, filed Oct. 14, 1997, now U.S. Pat. No. 5,985,848, thecontents of which are hereby incorporated by reference in theirentirety.

TECHNICAL FIELD

The invention relates to certain nucleoside analogues, the use of thesecompounds as pharmaceuticals, pharmaceutical compositions containing thecompounds and processes for preparing the compounds.

BACKGROUND OF THE INVENTION

Purine nucleoside phosphorylase (PNP) catalyses the phosphorolyticcleavage of ribo- and deoxyribonucleosides, for example, those ofguanine and hypoxanthine to give the corresponding sugar-1-phosphate andguanine, hypoxanthine, or other purine bases.

Humans deficient in purine nucleoside phosphorylase (PNP) suffer aspecific T-cell immunodeficiency due to an accumulation of dGTP and itstoxicity to stimulated T lymphocytes. Because of this, inhibitorsagainst PNP are immunosuppressive, and are active against T-cellmalignancies. Clinical trials are now in progress using9-(3-pyridylmethyl)-9-deazaguanine in topical form against psoriasis andin oral form for T-cell lymphoma and immunosuppression (BioCrystPharmaceuticals, Inc). The compound has an IC₅₀ of 35 nM for the enzyme.In animal studies, a 50 mg/kg oral dose is required for activity in acontact sensitivity ear swelling assay in mice. For human doses, thiswould mean approximately 3.5 grams for a 70 kg human. With thisinhibitor, PNP is difficult to inhibit due to the relatively highactivity of the enzyme in blood and mammalian tissues.

Nucleoside and deoxynucleoside hydrolases catalyse the hydrolysis ofnucleosides and deoxynucleosides. These enzymes are not found in mammalsbut are required for nucleoside salvage in some protozoan parasites.Purine phosphoribosyltransferases (PPRT) catalyze the transfer of purinebases to 5-phospho-α-D-ribose-1-pyrophosphate to form purine nucleotide5′-phosphates. Protozoan and other parasites contain PPRT which areinvolved in essential purine salvage pathways. Malignant tissues alsocontain PPRT. Some protozoan parasites contain purine nucleosidephosphorylases which also function in purine salvage pathways.Inhibitors of nucleoside hydrolases, purine nucleoside phosphorylasesand PPRT can be expected to interfere with the metabolism of parasitesand therefore be usefully employed against protozoan parasites.Inhibitors of PNP and PPRT can be expected to interfere with purinemetabolism in malignant tissues and therefore be usefully employedagainst malignant tissues.

It is an object of the invention to provide pharmaceuticals which arevery effective inhibitors of PNP, PPRT and/or nucleoside hydrolases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: FIG. 1 shows purine nucleoside phosphorylase activity with timeat a range of concentrations of the product of Example 1 (Compound Ib).

FIG. 2: FIG. 2 shows fitting of a purine nucleoside phosphorylaseactivity progress curve to the kinetic model.

FIG. 3: FIG. 3 shows K_(i)* determination by the curve fit method forCompound Ib inhibition of bovine purine nucleoside phosphorylase.

FIG. 4: FIG. 4 shows a progress curve for bovine purine nucleosidephosphorylase showing slow-onset inhibition by Compound Ib.

FIGS. 5A-5C: FIGS. 5A-5C shows the effect of oral administration ofCompound Ib on the PNP activity of mouse blood.

FIGS. 6A-6C: FIGS. 6A-6B shows the K_(i) determination for Compound Ibwith protozoan nucleoside hydrolase.

FIG. 7: FIG. 7 shows the progress curve for purinephosphoribosyltransferase showing slow-onset inhibition by the5′-phosphate of Compound Ib. Inhibition of the malaria enzyme.

FIG. 8: FIG. 8 shows the K₁* determination for the 5′-phosphate ofCompound Ib inhibition of human purine phosphoribosyltransferase.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the invention provides compounds having the formula:

wherein A is CH or N; B is chosen from OH, NH₂, NHR, H or halogen; D ischosen from OH, NH₂, NHR, H, halogen or SCH₃; R is an optionallysubstituted alkyl, aralkyl or aryl group; and X and Y are independentlyselected from H, OH or halogen except that when one of X and Y ishydroxy or halogen, the other is hydrogen; and Z is OH or, when X ishydroxy, Z is selected from hydrogen, halogen, hydroxy, SQ or OQ, Q isan optionally substituted alkyl, aralkyl or aryl group; or a tautomerthereof; or a pharmaceutically acceptable salt thereof; or an esterthereof; or a prodrug thereof.

Preferably when either of B and/or D is NHR, then R is C₁-C₄ alkyl.

Preferably when one or more halogens are present they are chosen fromchlorine and fluorine.

Preferably when Z is SQ or OQ, Q is C₁-C₅ alkyl or phenyl.

Preferably D is H, or when D is other than H, B is OH.

More preferably, B is OH, D is H, OH or NH₂, X is OH or H, Y is H, mostpreferably with Z as OH, H or methylthio, especially OH.

It will be appreciated that the representation of a compound of formula(I) wherein B and/or D is a hydroxy group used herein is of theenol-type tautomeric form of a corresponding amide, and this willlargely exist in the amide form. The use of the enol-type tautomericrepresentation is simply to allow fewer structural formulae to representthe compounds of the invention.

The present invention also provides compounds having the formula:

wherein A, X, Y, Z and R are defined for compounds of formula (I) wherefirst shown above; E is chosen from CO₂H or a corresponding salt form,CO₂R, CN, CONH₂, CONHR or CONR₂; and G is chosen from NH₂, NHCOR,NHCONHR or NHCSNHR; or a tautomer thereof, or a pharmaceuticallyacceptable salt thereof, or an ester thereof, or a prodrug thereof.

Preferably E is CONH₂ and G is NH₂.

More preferably, E is CONH₂, G is NH₂, X is OH or H, is H, mostpreferable with Z as OH, H or methylthio, especially OH.

Particularly preferred are the following compounds:

1.(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol

2.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol

3.(1R)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

4.(1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

5.(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol

6.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol

7.(1R)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erthro-pentitol

8.(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

9.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol

10.(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

11.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

12.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol

13.(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol

14.(1R)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

15.(1S)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

16.(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol

17.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol

18.(1R)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

19.(1S)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

20.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol

21.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitol

22.(1R)-1-C-(S-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

23.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

24.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol

25.(1S)-1-C-(3-amino-2-carboxamido-4-pyrroly)-1,4-dideoxy-1,4-imino-D-ribitol.

26.(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol5-phosphate

27.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol5-phosphate

28.(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-1,4-dideoxy-1,4-imino-D-ribitol

Most preferred are compounds Ib and Ic, their tautomers andpharmaceutically acceptable salts.

The biological availability of a compound of formula (I) or formula (Ia)can be enhanced by conversion into a pro-drug form. Such a pro-drug canhave improved lipophilicity relative to the compound of formula (I) orformula (Ia), and this can result in enhanced membrane permeability. Oneparticularly useful form of a pro-drug is an ester derivative. Itsutility relies upon the action of one or more of the ubiquitousintracellular lipases to catalyse the hydrolysis of these estergroup(s), to release the compound of formula (I) and formula (Ia) at ornear its site of action.

In one form of a prodrug, one or more of the hydroxy groups in acompound of formula (I) or formula (Ia) can be O-acylated, to make, forexample a 5-O-butyrate or a 2,3-di-O-butyrate derivative.

Prodrug forms of 5-phosphate ester derivative of a compounds of formula(I) or formula (Ia) can also be made and may be particularly useful,since the anionic nature of the 5-phosphate may limit its ability tocross cellular membranes. Conveniently, such a 5-phosphate derivativecan be converted to an uncharged bis(acyloxymethyl) ester derivative.The utility of such a pro-drug relies upon the action of one or more ofthe ubiquitous intracellular lipases to catalyse the hydrolysis of theseester group(s), releasing a molecule of formaldehyde and the compound offormula (I) or formula (Ia) at or near its site of action.

Specific examples of the utility of, and general methods for making,such acyloxymethyl ester pro-drug forms of phosphorylated carbohydratederivatives have been described, e.g. Kang et al., NucleosidesNucleotides 17 (1998) 1089; Jiang et al., J. Biol. Chem., 273 (1998)11017; Li et al., Tetrahedron 53 (1997) 12017; and Kruppa et al.,Bioorg. Med. Chem. Lett., 7 (1997) 945.

According to another aspect of the invention, there is provided apharmaceutical composition comprising a pharmaceutically effectiveamount of a compound of the first aspect of the invention.

Preferably the pharmaceutical composition comprises a compound chosenfrom the preferred compounds of the first aspect of the invention; morepreferably the compound is chosen from the more preferred compounds ofthe first aspect. Most preferably the compound is the compound offormula Ib or Ic.

In another aspect the invention provides methods for treatment ofdiseases or conditions in which it is desirable to decrease the level ofT lymphocyte activity. The methods comprise administering apharmaceutically effective dose of a compound of the invention to apatient requiring treatment.

The diseases include T-cell malignancies and autoimmune diseasesincluding arthritis and lupus. This aspect of the invention alsoincludes use of the compounds for immunosuppression for organtransplantation and for inflammatory disorders. The invention includesuse of the compounds for manufacture of medicaments for thesetreatments.

In another aspect the invention provides a method for treatment and/orprophylaxis of parasitic infections, particularly those caused byprotozoan parasites. Included among the protozoan parasites are those ofthe genera Giardia, Trichomonas, Leishmania, Trypanosoma, Crithidia,Herpetomonas, Leptomonas, Histomonas, Eimeria, Isopora and Plasmodium.An example of a parasitic infection caused by Plasmoodium is malaria.The method can be advantageously applied with any parasite containingone or more nucleoside hydrolases inhibited by the compound of theinvention when administered in an amount providing an effectiveconcentration of the compound at the location of the enzyme.

In another aspect, the invention provides a method of preparing thecompounds of the first aspect of the invention. The method may includeone or more of methods (A)-(Z) and (AA)-(AF).

Method (A): (4-hydroxypyrrolo[3,2-d]pyrimidines and access to 5′-deoxy-,5′-deoxy-5′-halogeno-, 5′-ether and 5′-thio-analogues)

reacting a compound of formula (II)

[wherein Z′ is a hydrogen or halogen atom, a group of formula SQ or OQ,or a trialkylsilyloxy, alkyldiarylsilyloxy or optionally substitutedtriarylmethoxy group and Q is an optionally substituted alkyl, aralkylor aryl group,] (typically Z′ is a tert-butyldimethylsilyloxy, trityloxyor similar group) sequentially with N-chlorosuccinimide then asterically hindered base (such as lithium tetramethylpiperadide) to forman imine, then with the anion of acetonitrile (typically made bytreatment of acetonitrile with n-butyllithium) followed by di-tert-butyldicarbonate. This generates a compound of formula (III)

[wherein Z′ is as defined for formula (II) where first shown above]which is then elaborated following the approach used to prepare9-deazainosine [Lim et al., J. Org. Chem. , 48 (1983) 780] in which acompound of formula (III) is condensed with (Me₂N)₂CHOBu^(t) andhydrolyzed under weakly acidic conditions to a compound of formula (IV)

[wherein Z′ is as defined for formula (II) where first shown above]which is then sequentially condensed with a simple ester of glycine(e.g. ethyl glycinate) under mildly basic conditions, cyclized byreaction with a simple ester of chloroformic acid (e.g. benzylchloroformate or methyl chloroformate) and then deprotected on thepyrrole nitrogen by hydrogenolysis in the presence of a noble metalcatalyst (e.g. Pd/C) in the case of a benzyl group or under mildly basicconditions in the case of a simple alkyl group such as a methyl group,to give a compound of formula (V)

[wherein Z′ is as defined for formula (II) where first shown above, andR is an alkyl group] (typically R is a methyl or ethyl group) which isthen condensed with formamidine acetate to give a compound of formula(VI)

[wherein Z′ is as defined for formula (II) where first shown above]which is then fully deprotected under acidic conditions, e.g. bytreatment with trifluoroacetic acid.

Methods for the preparation of a compound of formula (II) wherein Z′ isa tert-butyldimethylsilyloxy group are detailed in Furneaux et al,Tetrahedron 53 (1997) 2915 and references therein.

A compound of formula (II) [wherein Z′ is a halogen atom], can beprepared from a compound of formula (II) [wherein Z′ is a hydroxygroup], by selective N-alkyl- or aralkyl-oxycarbonylation (typicallywith di-tert-butyl dicarbonate, benzyl chloroformate, or methylchloroformate and a base) or N-acylation (typically with trifluoroaceticanhydride and a base) to give a compound of formula (VII):

[wherein R is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group and Z′ is a hydroxy group]which is then either:

(i) 5-O-sulfonylated (typically with p-toluenesulfonyl chloride,methanesulfonyl chloride or trifluoromethanesulfonic anhydride and abase) to give a compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z′ is an optionally substituted alkyl- oraryl-sulfonyloxy group], then subjected to a sulfonate displacementreaction with a reagent capable of providing a nucleophilic source ofhalide ion (typically sodium, lithium or a tetraalkylammonium fluoride,chloride, bromide, or iodide); or

(ii) subjected to a reagent system capable of directly replacing aprimary hydroxy group with a halogen atom, for example as in theMitsunobu reaction (e.g. using triphenylphosphine, diethylazodicarboxylate and a nucleophilic source of halide ion as above), byreaction with diethylaminosulfur trifluoride (DAST), or by reaction withmethyltriphenoxyphosphonium iodide in dimethylformamide [see e.g.Stoeckler et al, Cancer Res., 46 (1986) 1774] or by reaction withthionyl chloride or bromide in a polar solvent such ashexamethylphosphoramide [Kitagawa and Ichino, Tetrahedron Lett., (1971)87] to give a compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z′ is a halogen atom], which is then selectivelyN-deprotected by acid- or alkali-catalyzed hydrolysis or alcoholysis orcatalytic hydrogenolysis as required for the N-protecting group in use.

A compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z′ is a hydroxy group] can also be prepared froma compound of formula (II) [wherein Z′ is a trialkylsilyloxy,alkyldiarylsilyloxy or optionally substituted triarylmethoxy group], byN-alkyl- or aralkyl-carboxylation or N-acylation as above, thenselective 5-O-deprotection by acid-catalyzed hydrolysis or alcoholysis,catalytic hydrogenolysis, or treatment with a source of fluoride ion (egtetrabutylammonium fluoride) as required for the 5-O-protecting group inuse.

The compound of formula (II) [wherein Z′ is a hydrogen atom] can beprepared from either:

(i) a 5-hydroxy compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z′ is a hydroxy group], by formation and radicaldeoxygenation of a 5-O-thioacyl derivative; or

(ii) a 5-deoxy-5-halogeno-compound of formula (VII) [wherein Z′ is achlorine, bromine or iodine atom] by reduction, either using a hydridereagent such as tributyltin hydride under free radical conditions, or bycatalytic hydrogenolysis, typically with hydrogen over a palladiumcatalyst; followed by selective N-deprotection by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the N-protecting group in use.

A compound of formula (II) [wherein Z′ is an optionally substitutedalkylthio, aralkylthio or arylthio group] can be prepared by reaction ofa 5-deoxy-5-halogeno or a 5-O-sulfonate derivative of formula (VII)[wherein R is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group and Z′ is a halogen atom or anoptionally substituted alkyl- or aryl-sulfonyloxy group] mentionedabove, with an alkali metal or tetraalkylammonium salt of thecorresponding optionally substituted alkylthiol, aralkylthiol orarylthiol followed by selective N-deprotection by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the N-protecting group in use [see e.g. Montgomery etal., J. Med. Chem., 17 (1974) 1197].

The compound of formula (II) (wherein Z′ is a group of formula OQ, and Qis an optionally substituted alkyl, aralkyl or aryl group] can beprepared from a 5-hydroxy compound of formula (VII) [wherein R is analkyl- or aralkyl-oxycarbonyl group or an optionally substituted alkyl-or aryl-carbonyl group and Z is a hydroxy group], by

(i) reaction with an alkyl or aralkyl halide in the presence of a base(e.g. methyl iodide and sodium hydride, or benzyl bromide and sodiumhydride, in tetrahydrofuran as solvent); or

(ii) sequential conversion to a 5-O-sulfonate derivative (as above) andreaction with an alkali metal or tetraalkylammonium salt of the desiredphenol, followed by selective N-deprotection by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the N-protecting group in use.

It will be appreciated that the conversions above are conventionalreactions employed in carbohydrate chemistry. Many alternative reagentsand reaction conditions can be employed that will effect theseconversions, and references to many of these can be found in theSpecialist Periodical Reports “Carbohydrate Chemistry”, Volumes 1-28,published by the Royal Society of Chemistry, particularly in thechapters on Halogeno-sugars, Amino-sugars, Thio-sugars, Esters,Deoxy-sugars, and Nucleosides.

Method (B): (2-amino-4-hydroxypyrrolo[3,2-d]pyrimidines)

reacting a compound of formula (V) [wherein Z′ is as defined for formula(II) where first shown above, and R is an alkyl group] with benzoylisothiocyanate then methyl iodide in the presence of a base (e.g. DBU orDBN) following the approach used to prepare 9-deazaguanosine and itsderivatives [see e.g. Montgomery et al., J. Med. Chem. 36 (1993) 55, Limet al., J. Org. Chem., 48 (1983) 780, and references therein] to give acompound of formula (VIII)

[wherein Z′ is a trialkylsilyloxy, alkyldiarylsilyloxy or optionallysubstituted triarylmethoxy group, a hydrogen or halogen atom, SQ or OQwherein Q is an optionally substituted alkyl, aralkyl or aryl group andR is an alkyl group] (typically Z′, when a protected hydroxy group, is atert-butyldimethylsilyloxy, trityloxy or similar group, and R is amethyl or ethyl group) which is then cyclized in the presence of ammoniato give a separable mixture of compounds of formula (IX)

[wherein D is an amino or methylthio group, and Z′ and R are as definedfor formula (VIII) where first shown above, or Z′ i s a hydroxy group](where for example a tert-butyldimethylsilyloxy group has been cleavedunder the reaction conditions) and the product of formula (IX) [whereinD is an amino or methylthio group] is fully deprotected under acidicconditions by the procedures set out in Method (A).

Method (C): (4-aminopyrrolo [3,2-d]pyrimidines)

reacting a compound of formula (IV) [wherein Z′ is as defined forformula (II) where first shown above] with aminoacetonitrile undermildly basic conditions, cyclization of the product by reaction with asimple ester of chloroformic acid (typically benzyl chloroformate ormethyl chloroformate) to give a compound of formula (X)

[wherein Z′ is a trialkylsilyloxy, alkyldiarylsilyloxy- or optionallysubstituted triarylmethoxy group, a hydrogen or halogen atom, SQ or OQwherein Q is an optionally substituted alkyl, aralkyl or aryl group andR is an aralkyl or alkyl group] (typically Z′, when a protected hydroxygroup, is a tert-butyldimethylsilyloxy, trityloxy or similar group, andR is a benzyl or methyl group) which is then deprotected on the pyrrolenitrogen by hydrogenolysis in the presence of a noble metal catalyst(e.g. Pd/C) in the case of a benzyl group or under mildly basicconditions in the case of a simple alkyl group such as a methyl group,and processed as described above for the transformation (V)→(VI)→(I) or(V)→(VIII)→(IX)→(I). This method follows the approach used to prepare9-deazaadenosine and its analogues [Lim and Klein, Tetrahedron Lett., 22(1981) 25, and Xiang et al., Nucleosides Nucleotides 15 (1996) 1821].

Method (D): (7-hydroxypyrazolo[4,3-d]pyrimidines—Daves' Methodology)

reacting a compound of formula (II) [as defined where first shown above]sequentially with N-chlorosuccinimide and a hindered base (such aslithium tetramethylpiperidide) to form an imine, then condensing thiswith the anion produced by abstraction of the bromine or iodine atomfrom a compound of formula (XIb) or (XIc)

[wherein R³ is a bromine or iodine atom and R⁴ is a tetrahydropyran-2-ylgroup] typically using butyllithium or magnesium, to give a productwhich is then fully deprotected under acidic conditions (as in Method(A)). Methods for preparing compounds of formula (XIb) and (XIc) andmixtures thereof are described in Zhang and Daves, J. Org. Chem., 57(1992) 4690, Stone et al., J. Org. Chem., 44 (1979) 505, and referencestherein.

It will be appreciated that while the tetrahydropyran-2-yl group isfavoured as the protecting group for this reaction, other O,N-protectinggroups can be used, and that this method will also be applicable to thesynthesis of analogous pyrazolo[4,3-d]pyrimidines bearing substituentsat position-5 and/or -7 of the pyrazolo[4,3-d]pyrimidine ringindependently chosen from a hydroxy group, an amino, alkylamino, oraralkylamino group or a hydrogen atom using analogues of compounds offormula (XIb) and (XIc) in which the ionizable hydrogen atoms of anyhydroxy or amino groups have been replaced by a suitable protectinggroups.

Method (E): (7-hydroxypyrazolo[4,3-d]pyrimidines—Yokoyama Method)

subjecting a 5-O-ether protected 2,3-O-isopropylidene-D-ribofuranosederivative, where the 5-ether substituent is typically a trialkylsilyl,alkyldiarylsilyl, an optionally substituted triarylmethyl or anoptionally substituted aralkyl group, particularly atert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl,trityl or benzyl group, to the following reaction sequence:

(i) condensation with the anion produced by abstraction of the bromineor iodine atom from a compound of formula (XIb) or (XIc) from Method(D);

(ii) oxidation of the resulting diol to a diketone, typically using aSwern oxidation or a variant thereof using a dimethylsulfoxide-basedoxidant (e.g. using a dimethylsulfoxide and trifluoroacetic anhydridereagent combination in dichloromethane solution at low temperature,typically −78° C., followed by triethylamine and warming to roomtemperature);

(iii) double reductive amination to form a1,4-dideoxy-1,4-imino-D-ribitol moiety, typically with sodiumcyanoborohydride and ammonium formate, ammonium acetate orbenzhydrylamine in methanol; and

(iv) removal of the protecting groups by acid-catalyzed hydrolysis (e.g.with 70% aqueous trifluoroacetic acid) and if required (as in the caseof the product made with benzhydrylamine or where an optionallysubstituted aralkyl group has been used for protecting the primaryhydroxyl group in the iminoribitol moiety) hydrogenolysis over a metalcatalyst (typically a palladium catalyst) or if desired (as in the caseof silyl ether protecting group) exposure to a reagent capable of actingas a source of fluoride ion, e.g. tetrabutylammonium fluoride intetrahydrofuran or ammonium fluoride in methanol). Conditions suitablefor effecting this sequence of reactions are reported in Yokoyama etal., J. Org. Chem., 61 (1996) 6079, and conditions for double reductiveamination with ammonium acetate or benzhydrylamine can be found inFurneaux et al., Tetrahedron 42 (1993) 9605 and references therein.

Method (F): (7-hydroxypyrazolo[4,3-d]pyrimidines—the Kalvoda Method)

reacting a compound of formula (II) [as defined where first shown above]sequentially with N-chlorosuccinimide and a hindered base (such aslithium tetramethylpiperadide) to form an imine, then with a combinationof trimethylsilyl cyanide and a Lewis acid (typically boron trifluoridediethyl etherate) followed by acid catalyzed hydrolysis to give acompound of formula (XII)

[wherein Z′ is a hydrogen or halogen atom, a hydroxy group, or a groupof formula SQ or OQ where Q is an optionally substituted alkyl, aralkylor aryl group] which is then converted by sequential selectiveN-protection (typically with trifluoroacetic anhydride, di-tert-butyldicarbonate, benzyl chloroformate, or methyl chloroformate and a base),and O-protection with an acyl chloride or anhydride and a base(typically acetic anhydride or benzoyl chloride in pyridine) to asuitably protected derivative of formula (XIII)

[wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group, Z′ is a hydrogen or a halogenatom, a group of formula SQ or OQ where Q is an optionally substitutedalkyl, aralkyl or aryl group, or a group of formula R²O, and R² is analkylcarbonyl or optionally substituted arylcarbonyl group] (typicallyR¹ will be a trifluoroacetyl, tert-butoxycarbonyl or benzyloxycarbonylgroup, and R² will be an acetyl or benzoyl group).

The carboxylic acid moiety in the resulting compound of formula (XIII)is then transformed into a pyrazolo[4,3-d]pyrimidin-7-one-3-yl moietyfollowing the method described by Kalvoda [Collect. Czech. Chem.Commun., 43 (1978) 1431], by the following sequence of reactions:

(i) chlorination of the carboxylic acid moiety to form an acyl chloride,typically with thionyl chloride with a catalytic amount ofdimethylformamide in an inert solvent;

(ii) use of the resulting acyl chloride to acylate hydrogen cyanide inthe presence of tert-butoxycarbonyltriphenylphosphorane (i.e.Ph₃P═CHCO₂Bu^(t)) to give a 3-cyano-2-propenoate derivative;

(iii) cycloaddition of this with diazoacetonitrile (which can beprepared from aminoacetonitrile hydrochloride and sodium nitrite) withconcomitant elimination of hydrogen cyanide to give a pyrazolederivative;

(iv) acid-catalyzed hydrolysis of the tert-butyl a ester in thispyrazole derivative to its equivalent carboxylic acid;

(v) Curtius reaction, typically with phenylphosphoryl azide and2,2,2-trichloroethanol in the presence of triethylamine, which convertsthe carboxylic acid moiety into a 2,2,2-trichloroethoxycarbonylaminogroup (i.e. the product is a carbamate);

(vi) reductive cleavage of this trichloroethyl carbamate, typically withzinc dust in methanol containing ammonium chloride;

(vii) condensation of the resulting ethyl4-amino-3-substituted-1H-pyrazole-5-carboxylate with formamidine acetateto give a compound of formula (XIV)

[wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group, Z′ is a hydrogen or a halogenatom, SQ or OQ where Q is an optionally substituted alkyl, aralkyl oraryl group, or a group of formula R²O, and R² is an alkylcarbonyl oroptionally substituted arylcarbonyl group, A is a nitrogen atom, B is ahydroxy group and D is a hydrogen atom] which is then—and O-deprotectedby acid- or alkali-catalyzed hydrolysis or alcoholysis or catalytichydrogenolysis as required for the O- and N-protecting groups in use.

Method (G): (7-aminopyrazolo[4,3-d]pyrimidines—the Buchanan Method)

reacting a compound of formula (II) [as defined where first shown above]sequentially with N-chlorosuccinimide and a hindered base (such aslithium tetramethylpiperadide) to form an imine, which is thentransformed into a 7-amino-pyrazolo[4,3-d]pyrimidine derivativefollowing the approach used to prepare formycin and its analogues byBuchanan and co-workers [J. Chem. Soc., Perkin Trans. I (1991) 1077 andreferences therein], by the following sequence of reactions:

(i) addition of 3,3-diethoxyprop-1-ynylmagnesium bromide or3,3-diethoxyprop-1-ynyllithium to the imine;

(ii) N-protection, typically with trifluoroacetic anhydride,di-tert-butyl dicarbonate, benzyl chloroformate, or methyl chloroformateand a base;

(iii) mild acid hydrolysis to remove the acid sensitive O-protectinggroups and convert the diethyl acetal moiety into an aldehydic moiety;

(iv) condensation with hydrazine to convert the 3-substitutedprop-2-ynal derivative into a 3-substituted pyrazole derivative;

(v) acylation, typically with acetic anhydride or benzoyl chloride inpyridine;

(vi) nitration, typically with ammonium nitrate, trifluoroaceticanhydride and trifluoroacetic acid, to produce an 3-substituted1,4-dinitopyrazole derivative;

(vii) reaction with a reagent capable of delivering cyanide ion,typically sodium cyanide in aqueous ethanol to cause a cine-substitutionof one of the two nitro-groups;

(viii) reduction of the residual nitro-group, typically with sodiumdithionite or by catalytic hydrogenation over a metal catalyst;

(ix) condensation with formamidine acetate to give a compound of formula(XIV) [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or anoptionally substituted alkyl- or aryl-carbonyl group, Z′ is a hydrogenor a halogen atom, SQ or OQ where Q is an optionally substituted alkyl,aralkyl or aryl group, or a group of formula R²O wherein R² is analkylcarbonyl or optionally substituted arylcarbonyl group, A is anitrogen atom, B is an amino group and D is a hydrogen atom] which isthen—and O-deprotected by acid- or alkali-catalyzed hydrolysis oralcoholysis or catalytic hydrogenolysis as required for the O- andN-protecting groups in use.

Method (H): (2′-deoxy-analogues)

effecting the overall 2′-deoxygenation of a compound of formula (I)[wherein X and Z are hydroxy groups, Y is a hydrogen atom, and A, B andD are as defined where this formula is first shown above] throughsequential:

(i) selective N-alkyl- or aralkyl-oxycarbonylation (typically withdi-tert-butyl dicarbonate, benzyl chloroformate, or methyl chloroformateand a base) or N-acylation (typically with trifluoroacetic anhydride anda base) of the 1,4-dideoxy-1,4-iminoribitol moiety in such a compound offormula (I); and

(ii) 3′,5′-O-protection of the resulting product by reaction with1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane and a base to give acompound of formula (XV):

[wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group, R² is either the same as R¹or is a hydrogen atom, and A, B and D are as defined for formula (I)where first shown above]

(iii) 2′-O-thioacylation of the resulting compound of formula (XV)(typically with phenoxythionocarbonyl chloride and a base; or sodiumhydride, carbon disulfide and methyl iodide);

(iv) Barton radical deoxygenation (typically with tributyltin hydrideand a radical initiator);

(v) cleavage of the silyl protecting group by a reagent capable ofacting as a source of fluoride ion, e.g. tetrabutylammonium fluoride intetrahydrofuran or ammonium fluoride in methanol; and

(vi) cleavage of the residual N- and O-protecting groups by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the protecting groups in use.

Reagents and reaction conditions suitable for conducting the key stepsin this transformation can be found in Robins et al., J. Am. Chem. Soc.,105 (1983) 4059; Solan and Rosowsky, Nucleosides Nucleotides 8 (1989)1369; and Upadhya et al., Nucleic Acid Res., 14(1986) 1747.

It will be appreciated that a compound of formula (I) has a nitrogenatom in its pyrrole or pyrazole ring capable of undergoing alkyl- oraralkyl-oxycarbonylation or acylation during step (i), or thioacylationduring step (ii), depending upon the reaction conditions employed.Should such derivatives be formed, the pyrrole or pyrazoleN-substituents in the resulting derivatives are either sufficientlylabile that they can be removed by mild acid- or alkali-catalyzedhydrolysis or alcoholysis, or do not interfere with the subsequentchemistry in the imino-ribitol moiety, and can be removed during thefinal deprotection step(s). If desired, this approach can be applied toa compound of formula (XV) [as defined above, but additionally bearingN-protecting groups on the. pyrazolo- or pyrrolo-pyrimidine moiety].Methods suitable for preparing such N-protected compounds can be foundin Ciszewski et al., Nucleosides Nucleotides 12 (1993) 487; andKambhampati et al., Nucleosides and Nucleotides 5 (1986) 539, as canmethods to effect their 2′-deoxygenation, and conditions suitable forN-deprotection.

Method (I): (2′-epi-analoques)

effecting the overall C-2′ epimerization of a compound of formula (I),by oxidizing and then reducing a compound of formula (XV) [as definedwhere first shown above] to give compound of formula (XVI):

[wherein R¹, R², A, B and D are as defined for formula (XV) where firstshown above] which may be present in a mixture with the starting alcoholof formula (XV), and then fully deprotecting this compound of formula(XVI) as set out in steps (v) and (vi) of Method (H).

Reagents and reaction conditions suitable for conducting the key stepsin this transformation can be found in Robins et al., Tetrahedron 53(1997) 447.

Method (J): (2′-deoxy-2′-halogeno- and2′-deoxy-2′-epi-2′-halogeno-analogues)

reacting compound of formula (XV) or (XVI) [as defined where first shownabove] by the methods set out in Method (A) for the preparation of acompound of formula (II) [wherein Z′ is a halogen atom] which involveeither:

(i) 2′-O-sulfonylation and sulfonate displacement with a halide ion; or

(ii) direct replacement of the 2′-hydroxy group with a halogen atom, e.gby the Mitsunobu reaction or reaction with diethylaminosulfurtrifluoride (DAST) to give a compound of inverted stereochemistry atC-2′, which is then fully deprotected as set out in steps (v) and (vi)of Method (H).

It will be appreciated that a compound of formula (XV) or (XVI) has anitrogen atom in its pyrrole or pyrazole ring capable of undergoingsulfonylation during step (i), depending upon the reaction conditionsemployed. Should such derivatives be formed, the pyrrole or pyrazoleN-sulfonate substituents in the resulting derivatives are eithersufficiently labile that they can be removed by mild acid- oralkali-catalyzed hydrolysis or alcoholysis, or do not interfere with thesubsequent chemistry in the iminoribitol moiety, and can be removedduring the final deprotection step(s).

If desired, this approach can be applied to a compound of formula (XV)or (XVI) [as defined above, but additionally bearing N-protecting groupson the pyrazolo- or pyrrolo-pyrimidine moiety]. Methods suitable forpreparing such N-protected compounds can be found in Ciszewski et al.,Nucleosides Nucleotides 12 (1993) 487; and Kambhampati et al.,Nucleosides and Nucleotides 5 (1986) 539, as can methods to effect2′-O-triflate formation and displacement by halide ion with inversion,and conditions suitable for N-deprotection.

Method (K): (5′-deoxy-, 5′-deoxy-5′-halogeno-, 5′-ether and5′-thio-analogues)

by applying the procedures described in Method (A) for converting acompound of formula (VII) [wherein R is an alkyl- or aralkyl-oxycarbonylgroup or an optionally substituted alkyl- or aryl-carbonyl group and Z′is a hydroxy group] into a compound of formula (II) [wherein Z′ is ahalogen or hydrogen atom or SQ or OQ where Q is an optionallysubstituted alkyl, aralkyl or aryl group alkylthio group of one to fivecarbon atoms] to a compound of formula (XVII):

[wherein R is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group, Z′ is a hydroxy group, and A,B and D are as defined for formula (I) where first shown above] which isthen fully deprotected under acidic conditions, e.g. by treatment withaqueous trifluoroacetic acid.

Such a compound of formula (XVII) can be prepared from a compound offormula (I) [wherein X and Z are both hydroxy groups, Y is a hydrogenatom and A, B, and D have the meanings defined for formula (I) wherefirst shown above] in the following two reaction steps, which may beapplied in either order:

(i) selective N-alkyl- or aralkyl-oxycarbonylation (typically withdi-tert-butyl dicarbonate, benzyl chloroformate, or methyl chloroformateand a base) or N-acylation (typically with trifluoroacetic anhydride anda base) of the 1,4-dideoxy-1,4-iminoribitol moiety; and

(ii) 2′,3′-O-isopropylidenation, which may be effected with a variety ofreagents, e.g. acetone and anhydrous copper sulfate with or withoutadded sulfuric acid; acetone and sulfuric acid; 2,2-dimethoxypropane andan acid catalyst; or 2-methoxypropene and an acid catalyst.

It will be appreciated that such a compound of formula (I) or formula(XVII) has a nitrogen atom in its pyrrole or pyrazole ring capable ofundergoing sulfonylation, thioacylation, acylation oraralkyl-oxycarbonylation, depending upon the reaction conditionsemployed. Should such derivatives be formed, the pyrrole or pyrazoleN-substituents in the resulting derivatives are either sufficientlylabile that they can be removed by mild acid- or alkali-catalyzedhydrolysis or alcoholysis, or do not interfere with the subsequentchemistry in the iminoribitol moiety, and can be removed during thefinal deprotection step(s).

Method (L): (2- and 4-aminopyrrolo[3,2-d]pyrimidine and 5-and7-aminopyrazolo[4,3-d]pyrimidine analogues)

chlorinating a compound of formula (XVIII)

[wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group, R² is an alkylcarbonyl oroptionally substituted arylcarbonyl group, X and Y are independentlychosen from a hydrogen or halogen atom, or a group of formula R²O,except that when one of X or Y is a halogen atom or a group of formulaR²O, the other is a hydrogen atom, Z′ is a group of formula R²O or, whenX is a group of formula R²O, Z′ is a hydrogen or halogen atom, a groupof formula R²O or of formula OQ or SQ wherein Q is an optionallysubstituted alkyl, aralkyl or an aryl group, A is a nitrogen atom or amethane group, and one of B or D is a hydroxy group, and the other is achlorine, bromine or hydrogen atom] with a chlorinating reagent, andthen displacing the chlorine atom with a nitrogen nucleophile by one ofthe following methods:

(i) ammoniolysis, typically using liquid ammonia, concentrated aqueousammonia, or a solution of ammonia in an alcohol such as methanol; or

(ii) conversion first to a triazole derivative, by addition of4-chlorophenyl phosphorodichloridate to a solution of the chloride and1,2,4-triazole in pyridine, and alkaline hydrolysis of both thetetrazole moiety and the ester protecting groups with ammoniumhydroxide;

(iii) reaction with a source of azide ion, e.g. an alkali metal azide ortetraalkylammonium azide, and reduction of the resulting product,typically by catalytic hydrogenation; or

(iv) reaction with an alkylamine or aralkylamine, such as methylamine orbenzylamine in methanol.

These conditions are sufficiently basic that O-ester groups willgenerally be cleaved but any residual O- or N-protecting groups can thenbe removed by acid- or alkali-catalyzed hydrolysis or alcoholysis orcatalytic hydrogenolysis as required for the protecting groups in use.

Suitable chlorinating agents are thionyl chloride—dimethylformamidecomplex [Ikehara and Uno, Chem. Pharm. Bull., 13 (1965) 221],triphenylphosphine in carbon tetrachloride and dichloromethane with orwithout added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) [De Napoli etal., J. Chem. Soc., Perkin Trans.1 (1995) 15 and references therein],phosphoryl chloride [Imai, Chem. Pharm. Bull., 12 (1964) 1030], orphenylphosphoryl chloride and sodium hydride.

Suitable conditions for such an ammoniolysis or a reaction with analkylamine can be found in Ikehara and Uno, Chem. Pharm. Bull., 13(1965) 221; Robins and Tripp, Biochemistry 12 (1973) 2179; Marumoto etal., Chem. Pharm. Bull., 23 (1975) 759; and Hutchinson et al., J. Med.Chem., 33 (1990) 1919].

Suitable conditions for conversion of a such a chloride to an amine viaa tetrazole derivative can be found in Lin et al., Tetrahedron 51 (1995)1055.

Suitable conditions for reaction with azide ion followed by reductioncan be found in Marumoto et al., Chem. Pharm. Bull., 23 (1975) 759.

Such a compound of formula (XVIII) can be prepared from a compound offormula (I) by selective N-alkyl- or aralkyl-oxycarbonylation (typicallywith di-tert-butyl dicarbonate, benzyl chloroformate, or methylchloroformate and a base) or N-acylation of the1,4-dideoxy-1,4-iminoribitol moiety and then O-acylation (typically withacetic anhydride or benzoyl chloride in pyridine). It will beappreciated that such a compound of formula (I) has a nitrogen atom inits pyrrole or pyrazole ring capable of undergoing alkyl- oraralkyl-oxycarbonylation or acylation depending upon the reactionconditions employed. Should such derivatives be formed, the pyrrole orpyrazole N-substituents in the resulting derivatives are eithersufficiently labile that they can be removed by mild acid- oralkali-catalyzed hydrolysis or alcoholysis, or do not interfere with thesubsequent chemistry, and can be removed during the final deprotectionstep(s).

The above chlorination—amination—deprotection sequence can also beapplied to a compound of formula (XVI:) [wherein B is a hydroxy group, Dis a hydrogen atom, Z′ is a hydrogen or halogen atom, or a group offormula R²O, R² is a trialkylsilyloxy or alkyldibrylsilyloxy group, oran optionally substituted triarylmethoxy, alkylcarbonyl or arylcarbonylgroup, R and A are as defined for formula (XVII) where first shownabove]. Suitable conditions for conducting this reaction sequence can befound in Ikehara et al., Chem. Pharm. Bull., 12 (1964) 267.

Method (M): (2,4-dihydroxypyrrolo [3,2-d]pyrimidine and5,7-dihydroxypyrazolo[4,3-d]pyrimidine analogues)

oxidation of either:

(i) a compound of formula (XVIII) [wherein R² is a hydrogen atom; X andY are independently chosen from a hydrogen or halogen atom, or a hydroxygroup, except that when one of X or Y is a halogen atom or a hydroxygroup, the other is a hydrogen atom; Z′ is a hydroxy group or, when X isa hydroxy group, Z′ is a hydrogen or halogen atom, a hydroxy group, orOQ; Q is an optionally substituted alkyl, aralkyl or aryl group; B is ahydroxy group or an amino group; D is a hydrogen atom; and R¹ and A areas defined for formula (XVIII) where first shown above] with bromine inwater; or

(ii) a compound of formula (XVIII) [wherein Z′ is a hydrogen or ahalogen atom, or a group of formula R²O, or OQ; Q is an optionallysubstituted alkyl, aralkyl or aryl group; B is a hydroxy group or anamino group, D is a hydrogen atom and R¹, R², X, Y and A are as definedfor formula (XVIII) where first shown above], with bromine or potassiumpermanganate in water or in an aqueous solvent mixture containing aninert, water-miscible solvent to improve the solubility of thesubstrate, to give a related compound of formula (XVIII) [but wherein Band D are now hydroxy groups], and then removal of any O- andN-protecting groups by acid- or alkali-catalyzed hydrolysis oralcoholysis or catalytic hydrogenolysis as required for the protectinggroups in use.

Such a compound of formula (XVIII) required for step (i) above can beprepared from a compound of formula (I) [wherein Z is Z′, and X, Y, Z,A, B and D are as defined for the required compound of formula (XVIII)]by selective N-alkyl- or aralkyl-oxycarbonylation (typically withdi-tert-butyl dicarbonate, benzyl chloroformate, or methyl chloroformateand a base) or N-acylation (typically with trifluoroacetic anhydride anda base) of the 1,4-dideoxy-1,4-iminoribitol moiety. This can then beconverted to the corresponding compound of formula (XVIII) required forstep (ii) above by O-acylation (typically with acetic anhydride orbenzoyl chloride in pyridine). It will be appreciated that such acompound of formula (I) has a nitrogen atom in its pyrrole or pyrazolering capable of undergoing alkyl- or aralkyl-oxycarbonylation oracylation depending upon the reaction conditions employed. Should suchderivatives be formed, the pyrrole or pyrazole N-substituents in theresulting derivatives are either sufficiently labile that they can beremoved by mild acid- or alkali-catalyzed hydrolysis or alcoholysis, ordo not interfere with the subsequent chemistry, and can be removedduring the final deprotection step(s).

Method (N): (4-amino-2-chloropyrrolo[3,2-d]pyrimidine and7-amino-5-chloropyrazolo[4,3-d]pyrimidine analogues)

chlorinating a compound of formula (XVIII) [wherein B and D are hydroxygroups and R¹, R², X, Y, Z′ and A are as defined for formula (XVIII)where first shown above] to give a corresponding dichloro-derivative offormula (XVIII) [wherein B and D are chlorine atoms], typically withneat phosphorous oxychloride, and then displacing the more reactivechloro-substituent selectively by ammoniolysis, typically usinganhydrous liquid ammonia in a pressure bomb or methanolic ammonia, whichsimultaneously cleaves the O-ester protecting groups. The residualN-protecting group is then removed by acid-catalyzed hydrolysis oralcoholysis or catalytic hydrogenolysis as required for the protectinggroups in use, to give a compound of formula (I) [wherein B is anamino-group and D is a chlorine atom].

The above dichloro-derivative of formula (XVIII) can be converted into acompound of formula (I) [wherein B and D are chlorine atoms] by removalof the O- and N-protecting groups by acid- or alkali-catalyzedhydrolysis or alcoholysis as required for the protecting groups in use.It will be appreciated that one of the chlorine atoms in theaforementioned compound of formula (XVIII) or of formula (I) is quitereactive and that conditions chosen for deprotection must be mild enoughthat they limit unwanted reactions involving this atom.

Suitable reaction conditions for the key steps in this method can befound in Upadhya et al., Nucleic Acid Res., 14 (1986) 1747 and Kitagawaet al., J. Med. Chem., 16 (1973) 1381.

Method (O): (2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues fromdichloro-compounds)

hydrolysis of a compound of formula (XVIII) [wherein B and D arechlorine atoms] available as an intermediate from the first reaction ofMethod (N), typically with aqueous potassium hydroxide or sodiumcarbonate, in the presence of an inert, water-miscible solvent such asdioxane to enhance solubility as required, followed by removal of theresidual N-protecting group by acid-catalyzed hydrolysis or alcoholysisor catalytic hydrogenolysis as required for the protecting groups inuse, to give a compound of formula (I) [wherein B is a hydroxy group andD is a chlorine atom].

Method (P): (2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues fromaminochloro-compounds)

deamination of a compound of formula (XVIII) [wherein B is an aminogroup, D is a chlorine atom, R¹ is an alkyl- or aralkyl-oxycarbonylgroup or an optionally substituted alkyl- or aryl-carbonyl group, R² isa hydrogen atom, Z′=Z and X, Y, Z and A are as defined for formula (I)where first shown above), available as an intermediate following thechlorination and ammonyolysis reactions of Method (N), by reaction withnitrosyl chloride, followed by removal of the protecting groups as setout in Method (N). Typical reaction conditions can be found in Sanghviet al., Nucleosides Nucleotides 10 (1991) 1417.

Method (Q): (4-halogenopyrrolo[3,2-d]pyrimidine and7-halogenopyrazolo[4,3-d]pyrimidine analogues)

reacting a compound of formula (XVIII) (wherein R¹ istert-butoxycarbonyl group, B is a hydroxy group, D is a hydrogen atomand R², X, Y, Z′ and A are as defined for formula (XVIII) where firstshown above] by a method used to prepare halogeno-formycin analogues[Watanabe et al., J. Antibiotic, Ser. A 19 (1966) 93] which involvessequential treatment with:

(i) phosphorous pentasulfide by heating in pyridine and water underreflux to give a mercapto-derivative;

(ii) methyl iodide to give a methylthio-derivative;

(iii) a base in a simple alcohol or an aqueous solution of a simplealcohol, e.g. sodium methoxide in methanol, to remove the O-protectinggroups; and

(iv) chlorine, bromine or iodine in absolute methanol to give ahalogeno-derivative which is then N-deprotected by reaction with aqueousacid, typically a concentrated trifluoroacetic acid solution.

Method (R): (pyrrolo[3,2-d]pyrimidine and pyrazolo[4,3-d]pyrimidineanalogues)

hydrogenolytic cleavage of the chloride intermediate resulting from thechlorination reaction used as the first reaction in Method (L), or thechloride intermediate resulting from the chlorination reaction step (iv)in Method (Q), or the compound of formula (I) produced by Method (Q),typically using hydrogen over palladium on charcoal as the catalyst,optionally with magnesium oxide present to neutralize released acid,followed by cleavage of any residual O- or N-protecting groups by acid-or alkali-catalyzed hydrolysis or alcoholysis as required for theprotecting groups in use.

Method (S): (N-alkylated 4-aminopyrrolo[3,2-d]pyrimidine and7-aminopyrazolo[4,3-d]pyrimidine analogues)

heating an O-deprotected methylthio-derivative produced by step (iii) ofMethod (Q) with an amine, e.g. methylamine, in absolute methanol in asealed tube or bomb, and then removing the N-protecting group byreaction with aqueous acid, typically a concentrated trifluoroaceticacid solution. This method has been used to prepare N-alkylated-formycinanalogues [Watanabe et al., J. Antibiotic, Ser. A 19 (1966) 93]; orreacting a compound of formula (I) [wherein either B or D is an aminogroup] with 1,2-bis[(dimethylamino)methylene]hydrazine andtrimethylsilyl chloride in toluene to convert the amino group into a1,3,4-triazole group, hydrolysis to cleave the O-silyl groups (e.g. withacetic acid in aqueous acetonitrile), and displacement of the1,3,4-triazole group with an alkylamine in a polar solvent (e.g. wateror aqueous pyridine). This method has been used to prepareN,N-dimethyl-formycin A [Miles et al., J. Am. Chem. Soc., 117 (1995)5951]; or subjecting a compound of formula (I) [wherein either B or D isan amino group] to an exchange reaction by heating it with an excess ofan alkylamine. This method has been used to prepare N-alkyl-formycin Aderivatives [Hecht et al., J. Biol. Chem., 250 (1975) 7343].

Method T: (2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues)

Selective chlorination of dihydroxy compound of formula (XVIII) [whereinB and D are hydroxy groups, and R¹, R², X, Y, Z′ and A are as definedfor formula (XVIII) where first shown above], taking advantage of thegreater reactivity of the 4-hydroxy group on a2,4-dihydroxypyrrolo[3,2-d]pyrimidine derivative and the 7-hydroxy groupon a 5,7-dihydroxypyrazolo[4,3-d]pyrimidine derivative, followed byremoval of protecting groups, using the methods set out in Method (N).

Method U: (2-halogeno-, 4-halogeno- and2,4-dihalogeno-pyrrolo[3,2-d]pyrimidine and 5-halogeno-, 7-halogeno-,and 5,7-dihalogeno-pyrazolo[4,3-d]pyrimidine analogues) diazotization ofa compound of formula (XVIII) [wherein one of B or D is an amino group,and the other is independently chosen from an amino group, or a halogenoor hydrogen atom, and R¹ , R² , X, Y, Z′ and A are as defined forformula (XVIII) where first shown above] and subsequent reaction usingone of the following procedures:

(i) with nitrous acid (made in situ from sodium nitrite) in the presenceof a source of halide ion. For replacement of an amino-group with afluorine atom, a concentrated aqueous solution of fluoroboric acid[Gerster and Robins, J. Org. Chem., 31 (1966) 3258; Montgomery andHewson, J. Org. Chem., 33 (1968) 432] or hydrogen fluoride and pyridineat low temperature (e.g. −25 to −30° C.) [Secrist et al., J. Med. Chem.,29 (1986) 2069] can serve both as the mineral acid and the fluoride ionsource; or

(ii) with an alkyl nitrite, typically tert-butyl or n-butyl nitrite, ina non-aqueous solvent in the presence of a source of halide ion. Forreplacement of an amino-group with a chlorine atom, a combination ofchlorine and cuprous chloride, or antimony trichloride can be used inchloroform as solvent [Niiya et al, J. Med. Chem., 35 (1992) 4557 andreferences therein]; or

(iii) with an alkyl nitrite, typically tert-butyl or n-butyl nitrite, ina non-aqueous solvent coupled with photohalogenation. For replacement ofan amino group with a chlorine, bromine or iodine atom, carbontetrachloride, bromoform, or diiodomethane have been used as reagent andsolvent and an incandescent light source (e.g. a 200 W bulb) has beenused to effect photohalogenation [Ford et al., J. Med. Chem., 38 (1995)1189; Driscoll et al., J. Med. Chem., 39 (1996) 1619; and referencestherein]; to give a corresponding compound of formula (XVIII) [wherein Bis a halogen atom and B is either a halogen atom or an amino group],followed by removal of the protecting groups as set out in Method (N).

The same transformations can be effected for a corresponding startingcompound of formula (XVIII) [wherein one of B or D is an amino group,and the other is a hydroxy group] if the hydroxy group is firstconverted to a thiol group [Gerster and Robins, J. Org. Chem., 31 (1966)3258]. This conversion can be effected by reaction with phosphorouspentasulfide by heating in pyridine and water under reflux (see Method(Q)).

Method (V): (4-iodo-pyrazolo[3,2-d]pyrimidine and7-iodopyrazolo[4,3-d]pyrimidine analogues)

treatment of corresponding chloro-analogue of formula (I) [wherein B isa chlorine atom] with concentrated aqueous hydroiodic acid, followingthe method of Gerster et al., J. Org. Chem., 28 (1963) 945.

Method (W): (5′-deoxy-5′-halogeno- and 5′-thio-analogues)

by reacting a compound of formula (XVIII) [wherein R² is a hydrogenatom; X and Y are independently chosen from a hydrogen or halogen atom,or a hydroxy group, except that when one of X or Y is a halogen atom ora hydroxy group, the other is a hydrogen atom; Z is a hydroxy group; andR¹, A, B and D are as defined for formula (XVIII) where first shownabove] with either

(i) a trisubstituted phosphine and a disulfide, e.g. tributylphosphineand diphenyl disulfide; or

(ii) a trisubstituted phosphine (e.g. triphenylphosphine) and carbontetrabromide; or

(iii) thionyl chloride or bromide.

and then removal of the N-protecting group by acid- or alkali-catalyzedhydrolysis or alcoholysis or catalytic hydrogenolysis as required forthe protecting group in use.

Conditions suitable for conducting such selective replacements of a5′-hydroxy group with a thio group or a halogen atom can be found inChern et al., J. Med. Chem., 36 (1993) 1024; and Chu et al., NucleosideNucleotides 5 (1986) 185.

Method (X): (5′-phospho-pyrazolo[3,2-d]pyrimidine and5′-phospho-pyrazolo[4,3-d]pyrimidine analogues)

reacting a compound of formula (XVII) [wherein R, Z′, A, B and D are asdefined where first shown) with

(i) a phosphitylation agent, such asN,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine, thenoxidizing the phosphite ester to a phosphate ester, e.g. with3-chloroperbenzoic acid; or

(ii) a phosphorylatiing agent, such as phosphoryl chloride ordibenzylchlorophosphate; and removing the protecting groups, e.g. byhydrogenolysis and treatment under acidic conditions as set out inMethod (A).

Method (Y): (3-aminopyrrole-2-carboxylic acid and4-amino-1H-pyrazole-5-carboxylic acid analogues)

fully deprotecting a compound of formula (V) as defined where firstshown, or an intermediate ethyl4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by step (vi) inMethod (F), by acid- or alkali-catalyzed hydrolysis or alcoholysis orcatalytic hydrogenolysis as required for the O- and N-protecting groupsin use.

Method (Z): (3-amino-2-cyanopyrroles and 4-amino-5-cyano-1H-pyrazoles)

fully deprotecting a compound of formula (X) as defined where firstshown above, or a 4-amino-5-cyanopyrazole intermediate produced by step(viii) in Method (G), by acid-or alkali-catalyzed hydrolysis oralcoholysis or catalytic hydrogenolysis as required for the O- andN-protecting groups in use.

Method (AA): (3-aminopyrrole-2-carboxamide and4-amino-1H-pyrazole-5-carboxamide analogues)

conversion of the cyano-group of a compound of formula (X) as definedwhere first shown above, or a 4-amino-5-cyano-1H-pyrazoles intermediateproduced by step (viii) in Method (G), into a carboxamido-group,conveniently by reaction with hydrogen peroxide and potassium carbonatein dimethylsulfoxide, and then fully deprotecting the resulting productby acid- or alkali-catalyzed hydrolysis or alcoholysis or catalytichydrogenolysis as required for the O- and N-protecting group in use.

Method (AB): (3-(thio)carbamoylpyrroles and4-(thio)carbamoyl-1H-pyrazoles)

reaction of a compound of formula (V) or formula (X) as defined wherefirst shown above, or a protected carboxamido-intermediate as preparedin Method (AA), or an intermediate ethyl4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by step (vi) inMethod (F), with an isocyanate or isothiocyanate of formula RNCO orRNCS, where R is as defined for compounds of formula (I) and then fullydeprotecting the resulting product by acid- or alkali-catalyzedhydrolysis or alcoholysis or catalytic hydrogenolysis as required forthe O- and N-protecting groups in use.

Method (AC): (esters of 3-aminopyrrole-2-carboxylic acid and4-amino-1H-pyrazole-5-carboxylic acid analogues)

converting the carboxylic acid group of a compound of formula (Ia)wherein E is CO₂H into an ester, which can be accomplished by a numberof well known methods for esterification. Conveniently an ester can bemade by reaction of the carboxylic acid in acidic solution of thealcohol, e.g., ethanolic hydrogen chloride.

Method (AD): (3-acylaminopyrroles and 4-acylamino-1H-pyrazoles)

reaction of a compound of formula (V) or (X) as defined where firstshown above, or an intermediate ethyl4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by step (vi) inMethod (F), with an acylating agent, e.g. an acyl chloride such asbenzoyl chloride, acid anhydride such as acetic anhydride in thepresence of a base, such as triethylamine, potassium carbonate orpyridine, and then fully deprotecting the resulting product acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the O- and N-protecting groups in use.

Method (AE): (N-mono- and N,N-di-substituted3-amino-pyrrole-2-carboxamide and 4-amino-1H-pyrazole-5-carboxamideanalogues)

converting the carboxylic acid group of a compound of formula (Ia)wherein E is CO₂H into an amide. Conveniently an amide can be made bycarbodiimide induced condensation (e.g. withN,N-dicylcohexylcarbodiimide) of the carboxylic acid with a primary orsecondary amine.

Method (AF): (N-mono- and N,N-di-substituted3-amino-pyrrole-2-carboxamide and 4-amino-1H-pyrazole-5-carboxamideanalogues)

condensing a compound of formula (V) as defined where first shown, or anintermediate ethyl 4-amino-3-substituted-1H-pyrazole-5-carboxylateproduced by step (vi) in Method (F), with a primary or secondary amineand fully deprotecting the resulting product by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the O- and N-protecting groups in use.

It will be appreciated that the approaches outlined in Methods (H), (I),(J), (K) and (W) are equally applicable to the synthesis of compounds offormula (Ia) to give analogous variations in the 1,4-imino-pentitolmoiety.

Method (AG): (Acyloxymethyl Ester Prodrugs)

reacting a 5-phosphate ester of a compound of formula (I) or formula(Ia) with benzylchloroformate in the presence of a base, convenientlyaqueous sodium bicarbonate, to form an N-benzyloxycarbonyl derivative,reacting this derivative with an acyloxymethyl halide of formulaRCO₂CH₂X where R is an alkyl group such as methyl, ethyl, propyl ortert-butyl and X is chloride, bromide or iodide, in the presence of abase, to form the 5-phosphate bis(acyloxymethyl) ester. Suitableconditions for the formation of the acetoxymethyl esters, usingacetoxymethyl bromide and diisopropylethylamine in dimethylformamide,can be found in Kruppa et al, Bioorg. Med. Chem. Lett., 7 (1997) 945.

When desired, e.g. as when the aforementioned N-benzyloxycarbonylderivative is not sufficiently soluble in the reaction solvent, thisderivative may first be converted into the corresponding stannylintermediates, e.g. the bis(tributylstannyl) phosphate derivative byreaction with tributyltin methoxide in methanol, prior to reaction withthe acyloxymethyl halide in the presence of tetrabutylammonium bromide,following the method described by Kang et al., Nucleosides Nucleotides17 (1998) 1089.

It will be appreciated that the conversion of such a 5-phosphate groupto the corresponding bis(acyloxymethyl) ester can be accomplished byutilizing O- and or N-protected derivatives of compounds of formula (I)or formula (Ia) if desired, so long as the protecting groups cansubsequently be removed without the use of strongly acidic or stronglybasic conditions. Typically this requires the use of hydrogenolysisconditions for deprotection, so that O- and N-benzyl, -benzyloxymethylor -benzyloxycarbonyl groups are favoured.

Further Methods

Compounds of the invention may also be prepared by other methods as willbe apparent to those skilled in the art.

Further Aspects

The compounds of the invention are useful both in free base form and inthe form of salts. The term “pharmaceutically acceptable salts” isintended to apply to non-toxic salts derived from inorganic or organicacids including for example salts derived from the following acids-hydrochloric, sulfuric, phosphoric, acetic, lactic, fumaric, succinic,tartaric, gluconic, citric, methanesulphonic and p-toluenesulphonicacids.

The compounds of the invention are potent inhibitors of purinenucleoside phosphorylases, nucleoside hydrolases and/orphosphoribosyltransferases. For example, the IC₅₀ values for thecompounds of formula (Ib) and formula (Ic) are less than 0.1 nM for bothcalf spleen PNP and human red blood cell PNP. The examples below providefurther detail of the effectiveness of this inhibitor. Purine nucleosidephosphorylase inhibitory activity can be determined by the coupledxanthine oxidase method using inosine as the purine substrate (H. M.Kalckar, J.) Biol. Chem. 167 (1947) 429-443. Purinephosphoribosyltransferase activity is detected in the same assay usinginosine 5′-phosphate as the substrate. Slow onset inhibitor binding canbe determined using methods such as those described by Merkler et al.,Biochemistry 29 (1990) 8358-8364. Parasite nucleoside hydrolase activitymay be measured inter alia by methods disclosed in published PCTinternational patent application WO 97/31008 and the references citedtherein.

The potency of the inhibitors of the invention provides importantadvantages over the prior art because of the relatively high activity ofPNP in blood and mammalian tissue. As mentioned above the requireddosage of 9-(3-pyridylmethyl)-9-deazaguanine may be of the order of 3.5grams per dose for a human adult. The present invention provides theadvantage that considerably lower quantities of the compounds arerequired. This allows cost saving and may also reduce unwanted sideeffects.

The amount of active ingredient to be administered can vary widelyaccording to the nature of the patients and the nature and extent of thedisorder being treated. Typically the dosage for an adult human will bein the range less than 1 to 1000 milligrams, preferably 0.1 to 100milligrams. The active compound can be administered with a conventionalpharmaceutical carrier and may be administered orally, by injection ortopically.

The preferred route of administration is oral administration. Foradministration by this route the compounds can be formulated into solidor liquid preparations, eg tablets, capsules, powders, solutions,suspensions and dispersions. Such preparations are well known in the artas are other oral dosage forms not listed here. In a preferredembodiment the compounds of the invention are tableted with conventionaltablet bases such as lactose, sucrose and corn starch together with abinder, a disintegration agent and a lubricant. These exipients are wellknown in the art. The binder may be for example corn starch or gelatin,the disintegrating agent may be potato starch or alginic acid and thelubricant may be magnesium stearate. Other components such as colouringagents and flavouring agents may be included.

Liquid forms for use in the invention include carriers such as water andethanol, with or without other agents such as a pharmaceuticallyacceptable surfactant or suspending agent.

The compounds of the invention may also be administered by injection ina physiologically acceptable diluent such as water or saline. Thediluent may comprise one or more of other ingredients such as ethanol,propylene glycol, an oil or a pharmaceutically acceptably surfactant.

Compounds of the invention may be applied to skin or mucous membranes.They may be present as ingredients in creams, preferably including apharmaceutically acceptable solvent to assist passage through the skinor mucous membranes. Suitable cream bases are well known to thoseskilled in the art.

The compounds of the invention may be administered by means of sustainedrelease systems for example they may be incorporated into a slowlydissolving tablet or capsule containing a solid or porous or matrix formfrom a natural or synthetic polymer.

EXAMPLES

The following examples further illustrate practice of the invention.Ratios of solvents are by volume.

Example 1 Preparation of(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolExample 1.1.

A solution of5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(Furneaux et al, Tetrahedron 53 (1997) 2915 and references therein) (2.0g) in pentane (40 ml) was stirred with N-chlorosuccinimide (1.2 g) for1h. The solids and solvent were removed and the residue was dissolved indry tetrahydrofuran (40 ml) and cooled to −78° C. A solution of lithiumtetramethylpiperidide (25 ml, 0.4 M in tetrahydrofuran) was added slowlydropwise. The resulting solution was then added via cannula to asolution of lithiated acetonitrile [prepared by the dropwise addition ofacetonitrile (2.08 ml, 40 mmol) to a solution of butyl lithium (29.8 ml,41.8 mmol) in dry tetrahydrofuran (50 ml) at −78° C., followed bystirring for 45 min and then addition of tetramethylpiperidine (0.67 ml,4 mmol)] at −78° C. The reaction mixture was stirred for 15 min thenquenched with water and partitioned between water and chloroform. Theorganic phase was dried and concentrated, and then chromatographyafforded(1S)-5-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(1) (0.83 g).

Example 1.2

A solution of the product from Example 1.1 (0.80 g) in dichloromethane(20 ml) containing di-tert-butyldicarbonate (0.59 g) was stirred at roomtemperature for 16 h. The solution was concentrated and thenchromatography afforded(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(2) (0.89 g).

Example 1.3

To a solution of the product from Example 1.2 (0.88 g) inN,N-dimethylformamide (5 ml) was added tert-butoxybis(dimethylamine)methane (1.5 ml) and the solution was heated at 65-70°C. for 1 h. Toluene (20 ml) was added and the solution was washed (×3)with water, dried and concentrated to dryness. The residue was dissolvedin tetrahydrofuran/acetic acid/water (1:1:1 v/v/v, 40 ml) at roomtemperature. After 1.5 h chloroform (50 ml) was added and the mixturewas washed with water (×2), aqueous sodium bicarbonate, and then driedand evaporated to dryness. Chromatography of the residue gave(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-(1-cyano-2-hydroxyethenyl)-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(3) (0.68 g)

Example 1.4

Glycine hydrochloride ethyl ester (0.76 g) and sodium acetate (0.9 g)were added to a stirred solution of the product from Example 1.3 (0.51g) in methanol (10 ml). The mixture was stirred at room temperature for16 h and then concentrated to dryness. Chromatography of the residuegave the(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-[1-cyano-2-(ethoxycarbonylmethylamino)ethenyl]-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol (4) (0.48)g as a diastereomeric mixture.

Example 1.5

A solution of the product from Example 1.4 (0.28 g) in drydichloromethane (12 ml) containing 1,8-diazabicyclo[5.4.0]undec-7-ene(1.5 ml) and benzyl chloroformate (0.74 ml) was heated under reflux for8 h, then cooled and washed with dilute aqueous HCl, aqueous sodiumbicarbonate, dried and concentrated. Chromatography of the residueafforded(1S)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(5) (0.22 g).

Example 1.6

A solution of the product from Example 1.5 (0.22 g) in ethanol (10 ml)was stirred with 10% Pd/C (50 mg) in an atmosphere of hydrogen for 3 h.The solids and solvent were removed and the residue was dissolved inethanol (10 ml) containing formamidine acetate (0.40 g) and the solutionwas heated under reflux for 8 h. The solvent was removed andchromatography of the residue gave(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1-C-[4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-1,4-imino-2,3-O-isopropylidene-D-ribitol(6) (156 mg).

Example 1.7

A solution of the product from Example 1.6 (66 mg) in trifluoroaceticacid (3 ml) was allowed to stand at room temperature overnight. Thesolution was concentrated and a solution of the residue in water waswashed (×2) with chloroform and then evaporated. The residue wasdissolved in methanol and treated with Amberlyst A21 base resin untilthe solution was pH˜7. The solids and solvent were removed and theresidue was dissolved in water, treated with excess aqueous HCl and thenlyophilized. Trituration of the residue with ethanol gave(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol(7) hydrochloride salt as a white solid (25 mg). Recrystallised from 90%ethanol, the crystalline solid darkened but did not melt below 300° C.NMR (300 MHz, D₂O with DCl, δ ppm): ¹³C (relative to internal acetone at33.2 ppm) 58.1 (C-1′), 61.4 (C-5′), 68.8 (C-4′), 73.3 (C-3′), 76.7(C-2′), 107.5 (q), 121.4 (q), 133.5 (C-2), 135.0 (q), 148.0 (C-6) and155.4 (q); ¹H (relative to internal acetone at 2.20 ppm), 3.90 (H-4′),3.96 (m, H-5′,5″), 4.44 (dd, H-3′, J_(2′,3′) 5.4 Hz, J_(3′,4′) 3.2 Hz),4.71 (dd, J_(1′,2′) 9.0 Hz, H-2′), 5.00 (d, H-1′), 8.00 (s, H-6) and9.04 (s, H-2)

Example 2 Preparation of(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolExample 2.1

A solution of(1S)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4dideoxy 1,4-imino-2,3-O-isopropylidene-D-ribitol (Example 1.5) (0.87 g)in ethanol was stirred with 10% Pd/C (100 mg) in an atmosphere ofhydrogen for 1.5 h. The solids and solvent were removed to give aresidue (0.61 g). To a solution of a portion of this residue (0.12 g) indichloromethane (10 ml) at 0° C. was added a solution of benzoylisothiocyanate in dichloromethane (31 mL in 1 ml). After 0.5 h thesolution was warmed to room temperature and1,8-diazabicyclo[5.4.0]undec-7-ene (80 mL) and methyl iodide (100 mL)were added. After another 0.5 h the reaction solution was applieddirectly to a silica gel column and elution afforded 0.16 g of(1S)-1-C-[3-(N-benzoyl-S-methylisothiocarbamoyl)amino-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol.

Example 2.2

A solution of this S-methylisothiocarbamoylamino derivative, (0.20 g) inmethanol saturated with ammonia was heated in a sealed tube at 95° C.for 16 h. The solvent was removed and chromatography of the residueafforded(1S)-1-C-[2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-N-tert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol.

Example 2.3

A solution of this protected iminoribitol (64 mq) in trifluoroaceticacid was allowed to stand at room temperature for 16 h. The solvent wasremoved and a solution of the residue in aqueous methanol (1:1) wastreated with Amberlyst A21 base resin until the pH of the solution was˜7. The solids and solvent were removed and a solution of the residue inwater was treated with excess HCl and then concentrated to dryness.Trituration with ethanol gave(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolhydrochloride salt (24 mg), which darkened at ca. 260° C. but did notmelt below 300° C. NMR (300 MHz, D₂O with DCl, δ ppm): ¹³C (relative tointernal acetone at 33.1 ppm) 58.0 (C-1′), 61.4 (C-5′), 68.6 (C-4′),73.3 (C-3′), 76.3 (C-2′), 105.2 (q), 114.8 (q), 132.1 (C-6), 135.3 (q),153.4 (q) and 156.4 (q); ¹H (relative to internal acetone at 2.20 ppm)3.87 (m, H-4′), 3.94 (m, H-5′,5″), 4.40 (dd, J_(2′,3′) 5.0 Hz, J_(3′,4′)3.2 Hz, H-3′), 4.65 (dd, , J_(1′,2′) 9.1 Hz, H-2′), 4.86 (d, H-1′) and7.71 (s, H-6).

Examples 3-24

The following compounds may be prepared according to methods disclosedin the general description:

3.(1R)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 1 using Method (H)

4.(1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 1 using Method (K).

5.(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 1 using Method (K).

6.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolmay be prepared from the product of Examples 1 or 2 using Method (M).

7.(1R)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 6 using Method (H).

8.(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 6 using Method (K).

9.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 6 using Method (K).

10.(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 2 by Method (H).

11.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 2 by Method (K).

12.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 2 using Method (K).

13.(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitolmay be prepared by Methods (D), (E) and (F).

14.(1R)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 13 using Method (H).

15.(1S)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 13 using Method (K).

16.(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 13 using Method (K).

17.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitolmay be prepared from the product of Example 13 using Method (M).

18.(1R)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentiolmay be prepared from the product of Example 17 using Method (H).

19.(1S)-1-C-(5,7-dihydroxypyrazolo(4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 17 using Method (K).

20.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[(4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 17 using Method (K).

21.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitolmay be prepared using a variation of Method (D) in which the compound ofFormula XIb or XIc is replaced by a corresponding compound in which thehydrogen atom in position 5 is replaced by protected amino group.

22.(1R)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 21 using Method (H).

23.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 21 using Method (K).

24.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 21 using Method (K)

Example 25 Enzyme Inhibition Results Example 25.1

Inhibition of purine nucleoside phosphorylases. Enzyme assays wereconducted to assess the effectiveness of the products of Examples 1 and2 (compounds Ib and Ic respectively) as inhibitors of purine nucleosidephosphorylase. The assays used human RBC and calf spleen purinenucleoside phosphorylase (ex Sigma, 90% pure) with inosine as substrate,in the presence of phosphate buffer, with detection of releasedhypoxanthine using xanthine oxidase coupled reaction.

Materials. Inosine was obtained from Sigma. Xanthine oxidase (EC1.1.3.22, buttermilk), human erythrocyte (as a lyophilized powder) andbovine spleen (in 3.2 M ammonium sulfate) purine nucleosidephosphorylases (EC 2.4.2.1) were purchased from Sigma. Human purinenucleoside phosphorylases obtained as a powder was reconstituted in 100mM sodium phosphate buffer (pH 7.4) and rapidly frozen and stored at−80° C. Kinetic experiments were performed on a Uvikon 933 double beamultraviolet/visible spectrophotometer (Kontron Instruments, San Diego,Calif.).

Protein Concentrations. Protein concentrations for both isozymes weredetermined based on the quantative ultraviolet absorbance, usingE_(1cm)1%=9.64 at 280 nm [Stoelkler et al, Biochemistry, 32 (1978) 278]and a monomer moleculer weight of 32,000 [Williams et al, Nucleic AcidsRes. 12 (1984) 5779].

Enzyme Assay. Enzymes were assayed spectrophotometrically using thecoupled xanthine oxidase method [Kalckar, J. Biol. Chem. 167 (1947) 429;Kim et al, J. Biol. Chem., 243 (1968) 1763]. Formation of uric acid wasmonitored at 293 nm. A 40 mM inosine solution gave an absorbance changeof 0.523 units at 293 m, upon complete conversion of inosine to uricacid and ribose 1-phosphate. Unless otherwise noted, the standard assayreaction contained: inosine (500 μM), potassium phosphate (50 mM, pH7.5); xanthine oxidase (0.06 units) and purine nucleoside phosphorylasein a final volume of 1.0 mL.

One-Third-the-Sites Inhibition. Reaction mixtures of 6.7 nM bovinepurine nucleoside phosphorylase containing varying amounts of compoundIb were pre-incubated at 30° C. for 1 hour. Reactions were initiated byaddition of substrate (40 μM inosine, 3 times the K_(m) value) andassayed at 30° C. The reaction containing 0.6 nM inhibitor(concentration ratio of [compound Ib]/[purine nucleosidephosphorylase]=0.09) showed 29% inhibition, that containing 1 nMinhibitor ([compound Ib])/ [purine nucleoside phosphorylase]=0.15)showed 44%, whereas the reaction containing 3 nM inhibitor ([compoundIb]/purine nucleoside phosphorylase]=0.44) had a rate decrease of 96%,and that containing 6 nM inhibitor ([compound Ib])/[purine nucleosidephosphorylate]=87%) showed 99% inhibition. These interactions are shownin FIG. 1.

Purine nucleoside phosphorylase is known to be a homotrimer with acatalytic site on each of the three protein subunits [Stoelkler et al,Biochemistry 32, (1978) 278]. When the concentration of enzyme subunitsis 6.7 nM, 50% inhibition of purine nucleoside phosphorylase occurs atapproximately 1.1 nM. This result demonstrates that compound Ib bindstightly and that binding of compound Ib to one site of the trimericenzyme leads to complete inhibition.

Activity Recovery from the Complex of Purine Nucleoside Phosphorylasewith Compound Ib. Purine nucleoside phosphorylase (6.7 μM) andsufficient compound Ib (3 μM) to inhibit 96% of purine nucleosidephosphorylase activity were incubated at 30° C. for 1 hour. An aliquotof this solution was diluted 1000-fold into a buffered solution of 500μM inosine containing xanthine oxidase (0.06 units). The production ofuric acid was monitored over time and the progres curve was fit to thekinetic model of FIG. 2.

Dilution of inhibited purine nucleoside phosphorylase into a largevolume of solution without inhibitor provided the rate of release ofcompound Ib from inhibited purine nucleoside phosphorylase. Underconditions of the experiment in FIG. 2, the time to achieve the newenzyme-inhibitor equilibrium is 5000 sec, an indication of a slow,tight-binding inhibitor (Morrison and Walsh, Advances Enzymol. 61 (1988)201]. The rate contant k₆ is an estimate of the apparent first-orderrate constant for dissociation of the complex under these experimentalconditions and is 2.9×10⁻⁴ sec⁻¹ in this example.

Inhibitory Mechanism. Slow, tight-binding inhibitors generally followthe kinetic mechanism [Morrison and Walsh, Advances Enzymol. 61 (1988)201]:

where EI is a rapidly formed, initial collision complex of purinenucleoside phosphorylase (E) and compound Ib (I) that slowly isomerizesto a tighter complex EI*. Product formation curves are described by thefollowing integrated rate equation 1:

P=v _(s) t+(v _(o) −v _(s)) (1−e ^(−k t))/k  1

where P is the amount of product hypoxanthine (observed as uric acid inthe present assay system), t is time, v_(o) is the initial rate, v_(s)is the final steady-state rate and k is the overall (observed) rateconstant given by equation 2:

k=k6+k5[(I/K _(i))/(1+(S/K _(m))+(I/K _(i)))]  2

where K_(m) is the Michaelis complex for purine nucleosidephosphorylase, S is inosine concentration, I is the concentration ofcompound Ib and K_(i) is as described below. The rate of formation ofthe tightly bound complex is k5 and the rate of its dissociation is k6.K_(i), the inhibition constant for standard competitive inhibition(which influences v_(o)) and K_(l)*, the overall inhibition constant(which influences v_(s)), are defined as:

K _(i) =k4/k3

K _(i) *=K _(i) [k ₆/(k ₅ +k ₆)]

Determination of K_(i)*. K_(i)* was determined by measuring v_(s) forreactions at a range of inhibitor concentrations, plotting v_(s) vs [I]and fitting the curve to the competive inhibition equation 3:

v _(s) =V _(max) S/[K _(m)(1+I/K _(i)*)+S]  3

where v_(max) is the uninhibited reaction rate for purine nucleosidephosphorylase, and the remaining terms are described above. The resultof this analysis indicates an overall effective inhibition constant(K_(i)*) of 2.5±0.2×10⁻¹¹ M (25±2 pM) for compound Ib (FIG. 3).

Approximation of K_(i), k₅ and k₆. Calculation of K_(i) directly fromv_(o) and the competitive inhibition equation (above) is difficult forcompound Ib because v_(o) changes very little as a function of I atinhibitor concentrations which cause complete inhibition following slowonset. This result establishes that the initial dissociation constantK_(i) is much greater than the equilibrium dissociation constant K_(i)*.

Approximations of k₅ and K_(i) were calculated from k (values obtainedfrom curve fits of equation 1, FIG. 4) by using equation 2. Using theknowledge that k₆<<k₅[(I/K_(i))/(1+(A/K_(m))+(I/K_(i))], equation 2 canbe rearranged so that a double reciprocal plot of 1/k vs 1/[I] gives astraight line with y intercept=1/k₅ and x intercept of−(1/k₅)/[K_(i)/k₅)*(A/K_(m)))]. Substitution of these values intoequation 2 give an approximation for k₆. FIG. 4 demonstrates theslow-onset, tight-binding inhibition which occurs when a smallconcentration of enzyme (0.8 nM) competes for 200 nM compound Ib in thepresence of 500 μM inosine. Under these conditions the apparent firstorder rate constant for onset of inhibition in FIG. 4 was 26×10⁻⁴ sec⁻¹.

The result of FIG. 4 demonstrates that even at inosine concentrationsover 100 times that present in human serum or tissues, compound Ib cangive 99% inhibition of the enzyme after several minutes of slow-onsetinhibition. Based on analyses of experiments of the type shown in FIGS.1-4, the experimentally estimated dissociation constants and rates forthe bovine purine nucleoside phosphorylase with compound Ib are:

K _(m)=15 μM

K _(i)=19±4 nM

K _(i)*=25±2 pM

k ₅=1.4±0.2×10⁻² sec⁻¹

k ₆=1.8±0.5×10⁻⁵ sec−1

Inhibition of Human Purine Nucleoside Phosphorylase. Studies similar tothose described above for the interaction of bovine purine nucleosidephosphorylase were conducted with purine nucleoside phosphorylase (PNP)from human erythrocytes. The values for the overall inhibition constant,K_(i)*, for the interaction of human and bovine PNP with compound Ibare:

enzyme K_(i)*, compound Ib K_(i)*, compound Ic human PNP 72 ± 26 pM 29 ±8 pM bovine PNP 23 ± 5 pM  30 ± 6 pM

The compound Ic is a more efficient inhibitor for the human enzyme thancompound Ib, but compound Ib is slightly more efficient at inhibitingthe bovine enzyme. Compounds Ib and Ic are more efficient at inhibitingboth PNP enzymes than previously reported compounds.

Summary of Compounds Ib and Ic as Inhibitors of Purine NucleosidePhosphorylases. Inhibitors usually function by binding at everycatalytic site to cause functional inhibition in living organisms. Theone-third-the-sites inhibition and the slow-onset tight-bindinginhibition described above indicate that compounds Ib and Ic are verypotent inhibitors of purine nucleoside phosphorylases able to functionin the presence of a large excess of substrate.

The methods for the determination of the kinetic constants are given indetail in Merkler, D. J., Brenowitz, M., and Schramm, V. L. Biochemistry29 (1990) 8358-8364.

Example 25.2

Oral Availability and in vivo Efficacy of Compound Ib as a PNPInhibitor. A single oral dose of 10⁻⁷ mole of Compound Ib (27 μg) wasadministered with food to a young adult male mouse. Blood samples werecollected from the tail at times indicated in FIG. 5. Dilution of bloodinto saline containing 0.2% Triton X-100 (final concentration 0.15%)resulted in lysis of blood cells and release of enzyme. PNP activity wasmeasured with inosine and phosphate as substrates as indicated above.The results establish that Compound Ib is absorbed into the blood andtaken up by blood cells to cause PNP inhibition with a half-time (t_(½))of 14 minutes. Blood samples were taken for an extended time andanalyzed for PNP activity to determine the biological t_(½) for CompoundIb for inhibitors of blood PNP. The activity of blood PNP recovered witha t_(½) of 100 hours. These results establish that Compound Ib is orallyavailable and has an extended period of biological effectiveness. Thesetests establish that the compounds described herein have favorablepharmacological lifetimes.

Inhibition of Protozan Nucleoside Hydrolases by Compounds Ib and Ic.Protozan parasites use the hydrolysis of purine nucleosides such asinosine to provide purine bases such as hypoxanthine to provideessential precursors for RNA and DNA synthesis. Protozoan parasites arepurine auxotrophs. Using inhibition methods similar to those describedabove, a nucleoside hydrolase from Crithidia fasciculata [Parkin, et al,J Biol, Chem. 266 (1991) 20658] and a nucleoside hydrolase fromTrypanosoma brucei brucei [Parkin, J. Biol. Chem. (1996) 21713] weretested for inhibition by compounds Ib and Ic. The inhibition ofnucleoside hydrolase from C. fasciculata by Compound Ib is exemplifiedin FIG. 6. Similar studies indicated that Coumpound Ib and Ic arenanamolar inhibitors for nucleoside hydrolases from C. fasciculata andfrom T. brucei brucei. Compound Ic (A═CH, B═NH₂, D═H, X═OH, Y═H, Z═OH)is a nanamolar inhibitor of both enzymes and Compound Va (OR═NH₂, z′═OH,CO₂Bu═H or H₂, and the isopropylidine group removed to form two hydroxylgroups) is also a nanamolar inhibitor of both enzymes. The results aresummarised below.

K_(i) Values (nM) Compound Compound Compound Compound enzyme sourceIa^(a) Ib^(b) Ic^(b) Va^(b) nucleoside 42 ± 2 nM  40 nM   7 nM  3 nMhydrolase C. fasciculata nuceloside 24 ± 3 nM 108 nM 0.9 nM 23 nMhydrolase T. brucei brucei ^(a)the average of multiple determinationsand associated errors. ^(b)single determination of K_(i).

The inhibitors bind in direct competition with substrate, therefore theK_(i) inhibition constants are direct competitive inhibition values. Thecompounds provide sufficient inhibition to the purine nucleosidehydrolases to inhibit protozoan parasites at readily accessiblepharmacological doses.

The methods and materials used are as described in published PCTinternational application WO 97/31008 using p-nitrophenyl riboside assubstrate.

Example 25.3

Inhibition of Purine Phosphoribisyl Transferases (PPRT) by 5′-Phosphatesof Compounds Ib and Ic. Protozoan parasites, human tissues and tumorsuse PPRT for salvage of purine bases. Interruption of PPRT activity isexpected to disrupt purine metabolism in these systems.5′-phosphorylated Compounds I and Ic were anlyzed for inhibition of PPRTfrom human and malarial origins. The slow-onset inhibition curve for the5′-phosphate of Compound Ib with malaria PPRT is illustrated in FIG. 7.The K_(i)* determination for the 5′-phosphate of Compound Ib withmalarial PPRT is shown in FIG. 8. Analysis of both human and malarialenzymes with the 5′-phosphates of Compounds Ib and Ic are summarizedbelow.

enzyme Compound Ib-5′-phosphate Compound Ic-5′-phosphate source K_(i)K_(i)* K_(i) K_(i)* PPRT human 40 nM 3 nM 14 nM 8 nM PPRT 33 nM 3 nM 48nM slow onset malaria not observed

Full inhibition studies indicated that the inhibitors are competitivewith IMP. The nanamolar inhibition constants for both inhibitors withboth enzymes are readily accessible pharmacologic doses of theseinhibitors. It is anticipated that the nucleoside kinase activities ofhuman and/or parasitic organisms will convert one or more of thecompounds described herein to the respective 5′-phosphates. Thesecompounds thereby provide precursors for pharmacologic doses of the5′-phosphates for intracellular interruption of PPRT activity. Thecellular uptake of Compounds I and Ic have been documented with mice andwith human red cells.

Example 26 Tablet

4 grams of the product of Example 1 is mixed with 96 grams of lactoseand 96 grams of starch. After screening and mixing with 2 grams ofmagnesium stearate, the mixture is compressed to give 250 milligramtablets.

Example 27 Gelatin Capsule

Ten grams of the product of Example 1 is finely ground and mixed with 5grams of talc and 85 grams of finely ground lactose. The powder isfilled into hard gelatin capsules.

Example 28 Preparation of(1R)-1,2,4-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-erythro-pentitolExample 28.1

A solution of(1S)-5-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(1.93 g) in trifluoroacetic acid (20 ml) was allowed to stand at roomtemperature overnight. The solution was concentrated and a solution ofthe residue in water was washed (×2) with chloroform and then evaporatedto afford (1S)-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-D-ribitol (1.0 g)as the trifluoroacetic acid salt.

Example 28.2

A solution of the crude product from Example 3.1 (1.0 g) in methanol (20ml) containing di-tert-butyldicarbonate (2.09 g) was adjusted to neutralpH by the addition of triethylamine and stirred at room temperature for16 h. The solution was concentrated and then chromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-D-ribitol(0.80 g).

Example 28.3

1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (0.9 ml) was addeddropwise to a solution of the product from Example 3.2 (0.8 g) andimidazole (0.70 g) in N,N-dimethylformamide (10 ml) at 0° C. Theresulting solution was allowed to warm to room temperature, diluted withtoluene, washed with water (×3), dried, concentrated and thenchromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(1.4 g).

Example 28.4

A solution of the product from Example 3.3 (1.5 g) in toluene (20 ml)containing thiocarbonyldiimidazole (0.9 g) was stirred at 90° C. for 2h. The solution was concentrated and then chromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-2-O-[imidazole(thiocarbonyl)]-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(1.8 g).

Example 28.5

To a solution of the product from Example 28.4 (1.8 g) in toluene (50ml) was added tri-n-butyltin hydride (1.0 ml) and the solution washeated at 80° C. for 3 h. The solution was concentrated and thenchromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.74 g).

Example 28.6

To a solution of the product from Example 3.5 (0.74 g) inN,N-dimethylformamide (10 ml) was addedtert-butoxy-bis(dimethylamino)methane (1.5 ml) and the solution washeated at 65-70° C. for 1 h. Toluene (20 ml) was added and the solutionwas washed (×3) with water, dried and concentrated to dryness. Theresidue was dissolved in tetrahydrofuran/acetic acid/water (1:1:1 v/v/V,40 ml) at room temperature. After 1.5 h, chloroform (50 ml) was addedand the mixture was washed with water (×2), aqueous sodium bicarbonate,and then dried and evaporated to dryness. Chromatography of the residuegave(1R)-N-tert-butoxycarbonyl-1-C-(1-cyano-2-hydroxyethenyl)-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.68 g).

Example 28.7

Glycine hydrochloride ethyl ester (0.90 g) and sodium acetate (1.0 g)were added to a stirred solution of the product from Example 3.6 (0.68g) in methanol (10 ml). The mixture was stirred at room temperature for16 h and then concentrated to dryness. Chromatography of the residuegave the(1R)-N-tert-butoxycarbonyl-1-C-[1-cyano-2-(ethoxycarbonylmethylamino)ethenyl]-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.80 g) as a diastereomeric mixture.

Example 28.8

A solution of the product from Example 3.7 (0.80 g) in drydichloromethane (20 ml) containing 1,8-diazabicyclo[5.4.0]undec-7-ene(3.6 ml) and benzyl chloroformate (1.7 ml) was heated under refluxovernight, then cooled and washed with dilute aqueous HCl and thenaqueous sodium bicarbonate, dried and concentrated. Chromatography ofthe residue afforded(1R)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.70 g).

Example 28.9

A solution of the product from Example 28.8 (0.28 g) in ethanol (10 ml)was stirred with formamidine acetate (0.50 g) under reflux for 8 h. Thesolvent was removed and chromatography of the residue gave(1R)-N-tert-butoxycarbonyl-1,2,4-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxa-1,3-diyl)-D-erythro-pentitol(120 mg).

Example 28.10

A solution of the product from Example 28.9 (120 mg) in trifluoroaceticacid (2 ml) was allowed to stand at room temperature overnight. Thesolution was concentrated and a solution of the residue in water waswashed (×2) with chloroform and then evaporated. The residue wasdissolved in tetrahydrofuran and treated with tetrabutylammoniumfluoride trihydrate (200 mg) and stirred for 1 h. The solvent wasevaporated and chromatography gave a residue which was redissolved inmethanolic HCl. The resulting precipitate was filtered to afford(1R)-1,2,4-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-erythro-pentitolhydrochloride salt as a white solid (17 mg) which darkened but did notmelt below 300° C. NMR (300 MHz, D₂O, d ppm): ¹³C 38.8 (C-2′), 53.4(C-1′), 59.3 (C-5′), 69.1 (C-4′), 71.5 (C-3′), 107.6 (q), 118.6 (q),130.4 (C-2), 135.9 (q), 144.6 (C-6), and 153.7 (q); ¹H 2.69 (dd, J 14.3Hz, J 6.4 Hz, H-2′), 2.60 (ddd, J 14.3 Hz, J 12.2 Hz, J 5.7 Hz, H-2″),3.87 (m, 3H, H-4′, H-5′), 4.57 (m, 1H, H-3′), 5.26 (dd, 1H, J 12.1 Hz, J6.4 Hz, H-1′), 7.80 (s, H-6) and 8.65 (s, H-2). HRMS (MH⁺) calc. forC₁₁H₁₄N₄O₃: 251.1144; found: 251.1143.

Example 29 Preparation of(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,2,4-trideoxy-1,4-imino-D-erythro-pentitolExample 29.1

A solution of(1R)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(Example 28.8) (0.78 g) in ethanol (10 ml) was stirred with 10% Pd/C(100 mg) in an atmosphere of hydrogen for 1.5 h. The solids and solventwere removed to give a residue (0.62 g). To a solution of this residuein dichloromethane (10 ml) at 0° C. was added a solution (4.8 ml) ofbenzoyl isothiocyanate in dichloromethane (0.30 ml in 10 ml). After 0.5h, the solution was warmed to room temperature and1,8-diazabicyclo[5.4.0]undec-7-ene (0.32 ml) and methyl iodide (0.70 ml)were added. After another 0.5 h the reaction solution was applieddirectly to a silica gel column and elution afforded 0.67 g of(1R)-1-C-[3-(1-benzamido-1-methylthiomethyleneamino)-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol.

Example 29.2

A solution of the product from Example 29.1 (0.67 g) in methanolsaturated with ammonia (20 ml) was heated in a sealed tube at 105° C.for 16 h. The solvent was removed and chromatography of the residueafforded(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.30 g).

Example 29.3

A solution of the product from Example 29.2 (300 mg) in trifluoroaceticacid (5 ml) was allowed to stand at room temperature for 16 h. Thesolvent was removed and the residue was dissolved in tetrahydrofuran,treated with tetrabutylammonium fluoride trihydrate (200 mg) and stirredfor 1 h. The solvent was removed and the residue was dissolved inmethanol (5.0 ml) and acetyl chloride (0.75 ml) was added dropwise andthe reaction allowed to stand at room temperature for 16 h. The reactionwas diluted with ether (25 ml) and the resulting crystals were filteredto afford(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,2,4-trideoxy-1,4-imino-D-erythro-pentitolhydrochloride salt (89 mg), which did not melt below 300° C. NMR (300MHz, D₂O d ppm): ¹³C 38.8 (C-2′), 53.4 (C-1′), 59.3 (C-5′), 69.1 (C-4′),71.5 (C-3′), 107.6 (q), 118.6 (q), 130.4 (C-2), 135.9 (q), 144.6 (C-6),and 153.7 (q); ¹H 2.69 (dd, 1H, J 14.3 Hz, J 6.3 Hz, H-2′), 2.63 (ddd,1H, J 14.1 Hz, J 12.3 Hz, J 5.7 Hz, H-2″), 3.88 (m, 3H, H-4′, H-5′),4.55 (m, 1H, H-3′), 5.14 (dd, 1H, J 12.2 Hz, J 6.3 Hz, H-1′), and 7.63(s, H-6).

Example 30 Preparation of(1S)-1,4,5-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride salt Example 30.1

A solution of the product from Example 1.5 (0.45 g) in dichloromethane(10 ml) was treated with triethylamine (0.45 ml),4-dimethylaminopyridine (20 mg) and then methanesulfonyl chloride (0.1ml). The solution was stirred for 1 h and then washed with 2M aq HCl, aqbicarbonate and processed conventionally. The crude product wasdissolved in toluene (10 ml) containing tetrabutylammonium bromide (1.55g) and the solution was heated at 100° C. for 2 h. The cooled solutionwas washed with water, and processed to give, after chromatography,(1S)-1-C-(3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl)-N-tert-butoxycarbonyl-5-bromo-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.27 g).

Example 30.2

A solution of the product from Example 30.1 (0.27 g) in ethanol (10 ml)containing triethylamine (0.19 ml) was stirred with 20% Pd(OH)₂/C (0.1g) in a hydrogen atmosphere for 16 h. The solids and solvent wereremoved and chromatography afforded(1S)-1-C-(3-amino-2-ethoxycarbonyl-4-pyrrolyl)-N-tert-butoxycarbonyl-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.15 g).

Example 30.3

A solution of the product from Example 30.2 (75 mg) in ethanolcontaining formamidine acetate (0.15 g) was heated under reflux for 4 h.The solvent was removed and chromatography afforded(1S)-N-tert-butoxycarbonyl-1,4,5-trideoxy-1-C-[4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-1,4-imino-2,3-O-isopropylidene-D-ribitol(69 mg).

Example 30.4

The product from Example 30.3 (69 mg) was dissolved in trifluoroaceticacid (5 ml) and the solution was allowed to stand at room temperaturefor 16 h. The solvent was removed and a solution of the residue in 50%aqueous ethanol (10 ml) was treated with Amberlyst A21 base resin untilthe pH was ˜7. The solids and solvent were removed and the residue wastreated with excess aqueous HCl and lyophilized to give(1S)-1,4,5-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride salt (46 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 155.6(C), 147.1 (CH), 137.4 (C), 132.6 (CH), 121.0 (C), 108.2 (C), 76.5(C-3), 75.6 (C-2), 63.2 (C-4), 58.2 (C-1), 18.1 (C-5).

Example 31 Preparation of(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4,5-trideoxy-1,4-imino-D-ribitolhydrochloride salt. Example 31.1

A solution of benzoyl isothiocyanate (0.33 ml of 0.4 ml in 5 ml ofdichloromethane) was added to the product from Example 5.2 (75 mg) indichloromethane (5 ml) at 0° C. After 1 h,1,8-diazabicyclo[5.4.0]undec-7-ene (0.06 ml) and methyl iodide (0.1 ml)were added and the solution was stirred at room temperature for 1 h.Chromatography then afforded1(S)-1-C-[3-(1-benzamido-1-methylthio-methyleneamino)-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.10 g). A solution of this material in methanol (5 ml) saturated withammonia was heated in a sealed tube at 95° C. for 16 h and thenevaporated. Chromatography afforded(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(28 mg).

Example 31.2

The product from Example 31.1 (28 mg) was treated as for Example 30.4above to give(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4,5-trideoxy-1,4-imino-D-ribitolhydrochloride salt (16 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 156.5(C), 153.5 (C), 135.8 (C), 131.7 (CH), 114.9 (C), 105.6 (C), 76.7 (C-3),75.7 (C-2), 63.4 (C-4), 58.1 (C-1), 18.4 (C-5).

Example 32 Preparation of(1S)-1-C-(4-aminopyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolhydrochloride salt Example 32.1

A solution of the product from Example 1.3 (0.15 g) in methanol (5 ml)containing aminoacetonitrile (0.12 g) and sodium acetate (0.20 g) washeated under reflux for 4 h and then concentrated. Chromatographyafforded1(S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-[1-cyano-2-cyanomethylamino-ethenyl]-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.12 g) as a diastereomeric mixture. A solution of this material indichloromethane (10 ml) containing 1,8-diazabicyclo[5.4.0]undec-7-ene(0.7 ml) and benzyl chloroformate (0.33 ml) was heated under reflux for1 h. Conventional processing and chromatography afforded(1S)-1-C-(3-amino-1-N-benzyloxycarbonyl-2-cyano-4-pyrrolyl)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.125 g).

Example 32.2

A solution of the product from Example 32.1 (0.125 g) in ethanol (10 ml)was stirred in an atmosphere of hydrogen with 10% Pd/C (20 mg) for 0.5h. The solids were removed, formamidine acetate (0.21 g) was added tothe filtrate and the solution was heated under reflux for 16 h and thenconcentrated. Chromatography of the residue gave(1S)-1-C-(4-aminopyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(80 mg).

Example 32.3

The product from Example 32.2 (80 mg) was treated as for Example 30.4above to give(1S)-1-C-(4-aminopyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolhydrochloride salt (35 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 152.1(C), 146.2 (CH), 140.7 (C), 135.3 (CH), 115.4 (C), 107.7 (C), 76.0(C-2), 73.1 (C-3), 68.4 (C-4), 61.3 (C-5), 58.3 (C-1).

Example 33 Preparation of(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol5-phosphate bis-ammonium salt

The product from Example 2.2 (0.13 g) in dry acetonitrile (6 ml)containing tetrazole (0.105 g) was stirred at room temperature whileN,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine was addedslowly dropwise until t.l.c. indicated complete reaction, thenmeta-chloroperbenzoic acid (60 mg) was added followed by further smallquantities of the oxidant until t.l.c. indicated the initial product wasfully reacted. Chloroform was added and the solution was washed withaqueous sodium bicarbonate, dried and concentrated. Chromatographyafforded the phosphate ester (190 mg) which was stirred in ethanol (10ml) in an atmosphere of hydrogen with 10% Pd/C (80 mg) for 1 h. Thesolids and solvent were removed and the residue was dissolved intrifluoroacetic acid (5 ml) and allowed to stand at room temperature for16 h. The solution was concentrated by evaporation and the residue inwater was applied to a column of Amberlyst A15 acid resin. The columnwas washed with water and then with 2M aqueous ammonia to elute theproduct. Concentration and trituration of the residue with waterafforded(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol5-phosphate bis-ammonium salt (50 mg), referred to as the 5′-phosphateof compound Ib. ¹³C NMR (75 MHz, TFA-D, d ppm): 146.9 (C), 144.0 (C),127.0 (C), 124.5 (CH), 105.1 (C), 95.6 (C), 66.3 (CH), 64.0 (CH), 59.2(CH), 56.2 (CH₂), 50.2 (CH).

Example 34 Preparation of(1S)-1,4,5-trideoxy-5-fluoro-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride salt Example 34.1

To a solution of the product from Example 1.2 (1.48 g) intetrahydrofuran (10 ml) was added tetrabutylammonium fluoride (6 ml, 1Min THF). After 2 h the solution was evaporated and chromatography of theresidue afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(1.15 g). A solution of 0.84 g of this material in dichloromethane (20ml) containing triethylamine (1.0 ml) was stirred whilediethylaminosulfur trifluoride (0.36 ml) was added. After 2 h, methanol(1 ml) was added and the solution was evaporated. Chromatography gave(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4,5-trideoxy-5-fluoro-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.36 g).

Example 34.2

The product from Example 34.1 (0.36 g) was treated in the same manner asdescribed for examples 1.3 and then 1.4 and 1.5 above to give(1S)-1-C-(3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl)-N-tert-butoxycarbonyl-1,4,5-trideoxy-5-fluoro-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.23 g).

Example 34.3

The product from Example 34.2 (0.12 g) was treated as described forexamples 1.6 and then 1.7 above to give, after lypohilization,(1S)-1,4,5-trideoxy-5-fluoro-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride salt (43 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 146.8(CH), 132.6 (CH), 83.0 (J_(C,F) 169 Hz, C-5), 76.1 (C-2), 72.7 (C-3),66.4 (J_(C,F) 18 Hz, C-4), 59.0 (C-1).

Example 35(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-1,4-dideoxy-1,4-imino-D-ribitolExample 35.1

Hydrogen peroxide (0.5 ml) was added dropwise to a solution of theproduct from Example 32.1 (90 mg) and potassium carbonate (50 mg) indimethylsulfoxide (1.0 ml). The reaction was stirred for 10 minutes,diluted with water (50 ml), extracted with ethyl acetate (3×20 ml), andthe combined organic layers dried and concentrated. Chromatography ofthe resulting residue afforded(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(20 mg).

Example 35.2

A solution of the product from Example 35.1 (20 mg) in trifluoroaceticacid (1 ml) was allowed to stand at room temperature for 16 h. Thesolvent was removed and the residue in water (20 ml) was washed withdichloromethane (2×5 ml). The aqueous layer was evaporated andchromatography afforded(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-1,4-dideoxy-1,4-imino-D-ribitol(10 mg). NMR (300 MHz, D₂O): ¹³C 59.3 (C-4′), 64.0 (C-5′), 67.7 (C-1′),74.4 (C-3′), 77.6 (C-2′), 113.2 (q), 124.1 (C-5), 126.2 (q), 141.0 (q),and 168.7 (q). HRMS (MH⁺) calc. for C₁₀H₁₇N₄O₄: 257.12498; found:257.12535.

Example 36 Preparation of(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo-[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolExample 36.1

2,4-Dihydroxy-6-methyl-5-nitropyrimidine (G. N. Mitchell and R. L.McKee, J. Org. Chem., 1974, 39, 176-179) (20 g) was suspended inphosphoryl chloride (200 ml) containing N,N-diethylaniline (20 ml) andthe mixture was heated under reflux for 2 h. The black solution wasconcentrated to dryness and the residue was partitioned between water(600 ml) and ether (150 ml). The aqueous phase was further extractedwith ether (150 ml) and the combined organic phases were washed withaqueous sodium bicarbonate and processed conventionally to give2,4-dichloro-6-methyl-5-nitropyrimidine (23.1 g).

Example 36.2

To a solution of the product of Example 36.1 (17 g) in benzyl alcohol(80 ml) was added a 1.1 M solution of sodium benzylate in benzyl alcohol(199 ml). After 1 h at room temperature, ether (500 ml) was added andthe solution was washed with water. The organic phase was dried andconcentrated to dryness under high vacuum. The crude residue in dryN,N-dimethylformamide (100 ml) and N,N-dimethylformamide dimethyl acetal(25 ml) was heated at 100° C. for 3 h and then the solution wasconcentrated to dryness. Trituration of the residue with ethanol andfiltration afforded2,4-dibenzyloxy-6-(2-dimethylaminovinyl)-5-nitropyrimidine as an orangesolid (24.5 g)

Example 36.3

Zinc dust (30 g) was added to a solution of the product from Example36.2 (20 g) in acetic acid (300 ml) with cooling to control theexotherm. The resulting mixture was then stirred for 2 h, filtered, andthe filtrate was concentrated to dryness. The residue was partionedbetween chloroform and aqueous sodium bicarbonate, the organic layer wasdried and then concentrated to dryness to give a solid residue of2,4-dibenzyloxypyrrolo[3,2-d]pyrimidine (15.2 g)

Example 36.4

Sodium hydride (0.5 g, 60% dispersion in oil) was added to a solution ofthe product from example 36.3 (2.0 g) in tetrahydrofuran (40 ml)followed by tert-butyldimethylsilyl chloride (1.37 g) and the mixturewas stirred for 1 h. The reaction was quenched with dropwise addition ofwater and then partitioned between ether (100 ml) and water (150 ml).The organic phase was dried and concentrated to dryness. A solution ofthe residue in dichloromethane (40 ml) was stirred whileN-bromosuccinimide added slowly poriton-wise until t.l.c. analysisindicated complete conversion to a less polar product. The solution waswashed with water, aqueous sodium bicarbonate, dried and concentrated.Chromatography of the residue afforded2,4-dibenzyloxy-7-bromo-9-N-tert-butyldimethylsilylpyrrolo[3,2-d]pyrimidineas a white solid (1.8 g).

Example 36.5

An imine was prepared from5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.30 g) by N-chlorination with N-chlorosuccinimide followed byelimination of hydrogen chloride with lithium tetramethylpiperidide asdescribed in Example 1.1, but with the following modifications: (i) whenaddition of the solution of lithium tetramethylpiperidide was complete,petroleum ether was added and the solution was washed with water, driedand concentrated to dryness; (ii) the residue was chromatographed onsilica gel eluted with 0.2% triethylamine and 30% ethyl acetate inhexanes to afford the pure imine (0.215 g). A solution of this imine inether (2 ml) was added to a solution prepared by slow addition ofbutyllithium (1.4 M in hexanes) to a solution of the product fromExample 36.4 (0.786 g) in anisole (20 ml) and ether (30 ml) at −70° C.until t.l.c. analysis indicated lithium exchange with the startingmaterial was complete. The mixture was allowed to slowly warm to ˜15°C., and then was washed with water, dried and concentrated.Chromatography of the residue afforded(1S)-1-C-)2,4-dibenzyloxy-9-N-tert-butyldimethylsilylpyrrolo[3,2-d]pyrimidin-7-yl)-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.225 g).

Example 36.6

A solution of the product from Example 36.5 (0.10 g) in ethanol (5 ml)was stirred in a hydrogen atmosphere with 10% palladium on charcoal(0.05 g) for 2 h. The solids and solvent were removed and concentratedaqueous hydrochloric acid (1 ml) was added to a solution of the residuein methanol (5 ml). After standing overnight, the solution wasconcentrated to dryness and the residue was extracted with ether andthen triturated with ethanol and filtered to give(1S)-1,4-dideoxy-1-C-)2,4-dihydroxypyrrolo[3,2-d][pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride (0.025 g). ¹³C NMR (D₂O), δ (ppm): 159.8 (C), 155.8 (C),137.1 (C), 131.4 (CH), 114.2 (C), 104.1 (C), 76.2 (CH), 73.7 (CH), 68.5(CH) 61.6 (CH₂) and 58.5 (CH).

Example 37 Preparation of1,4-dideoxy-(1S)-1-C-(2,4-dihydroxypyrrolo-[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol5-phosphate bis-ammonium salt Example 37.1

A solution tetrabutylammonium fluoride (1 M, 0.5 ml) was added to asolution of the bis-silylated product from Example 36.5 (110 mg) intetrahydrofuran. After 2 h, the solution was diluted with toluene,washed with water (×2), dried, and evaporated to dryness. The resultingsyrup was dissolved in methanol and tert-butoxycarbonic anhydride (65mg) was added. After 30 min, the reaction mixture was concentrated todryness and subjected to chromatography to give(1S)-1-C-(2,4-dibenzyloxypyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(64 mg).

Example 37.2

The product for Example 37.2 (64 mg) was converted by the methoddetailed in Example 33 into,1,4-dideoxy-(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol5-phosphate bis-ammonium salt (11 mg); ¹³C-NMR (D₂O), δ (ppm): 156.0(C), 151.9 (C), 134.0 (C), 127.3 (CH), 110.9 (C), 102.8 (C), 75.1 (CH),70.4 (CH), 65.1 (CH), 61.9 (CH₂), and 54.5 (CH).

Aspects of the invention have been described by way of example only andit should be appreciated that modifications and additions thereto may bemade without departing from the scope of the invention.

What is claimed is:
 1. A pharmaceutical composition comprising(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol,or tautomer thereof, or a pharmaceutically acceptable salt thereof.
 2. Apharmaceutical composition comprising(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol,or tautomer thereof, or a pharmaceutically acceptable salt thereof.